Antiviral agents

ABSTRACT

A second-step virus binding receptor is found in nature on the surface of animal and plant cells. This receptor is thought to be needed for virus penetration into target cells. The second-step receptor has been found to bind a wide variety of viruses belonging to a number of different families. The second-step receptor and natural or synthetic substances which correspond to or are analogous to the binding epitope of the second-step receptor in that they are able to bind to a site on the virus which recognizes the binding epitope of the natural second-step receptor, are therefore indicated for the diagnosis, prophylaxis or treatment of viral infections.

This application is a continuation of Ser. No. 06/916,542, filed Nov. 5,1986, now patented, U.S. Pat. No. 4,859,769. Ser. No. 916,542 is thenational stage of PCT/DK86/00007, which has an international filing dateof Jan. 13, 1986. This date is the effective filing date of Ser. No.916,542 pursuant to 35 U.S.C. 120,363.

The present invention relates to the use of certain compounds withvirus-binding properties for the diagnosis, prophylaxis or treatment ofviral infections, as well as antiviral agents and pharmaceuticalcompositions comprising these compounds.

Despite the extensive damage to animals, including human beings, andplants caused by viruses, no general rational therapy has yet beendevised against viral infections comparable to, e.g., antibiotictreatment in the case of bacterial infections. Although, in certaincases, a prophylactic approach in the form of a vaccine causingimmunization and, in most instances, producing resistance tore-infection for life has successfully been employed, it has not alwaysbeen possible to develop a vaccine of a sufficiently broad applicabilityto be effective against a wide variety of strains of the same viralspecies; this, for instance, has been a problem with the influenzavaccines which have hitherto been devised, so that immunization hasoccurred against the specific strain on which the vaccine has beenbased, but not against other, closely related strains with slightlydifferent antigenic properties.

In recent years, increasing attention has been paid to the importance,for a variety of biological interactions, of so-called receptors.Receptors, which are often glycolipids or glycoproteins, that is,consist of a carbohydrate portion linked to a lipid or a protein, formintegral parts of the plasma membrane of animal and plant cells, beinglocated on the surface of the cell membrane of a wide variety of cells.Their function as specific receptors for a wide range of biologicalentities is extremely diverse. Due to their carbohydrate portion beingexposed on the surface of the cell membrane, they may have antigenicproperties or function as cell surface markers; they may conferstructural rigidity to the outer monolayer of the membrane lipidbilayer; they may form part of a system for cell-cell interaction andrecognition; or they may play a part in the interaction of the cell withbioactive factors such as bacterial toxins, glycoprotein hormones ormicroorganisms, anchoring these to the cell surface. For instance, it isknown that there is a connection between such receptors and bacterialinfections in that receptor analogues may be used to inhibit thebacterial adhesion necessary to effect an infection.

For a viral infection to become established, the viral genome has topenetrate into the host cell. For some membrane-enveloped (having amembrane around the nucleocapside) viruses, this process is known toinvolve a two-step mechanism, namely the sequential attachment to andpenetration into the host cell (see reference 1; the list of referencesis given below in the section entitled "Bibliography"). It is known thatthe attachment is due to a receptor located on the surface of targetcells for viral infection (see reference 3). It has commonly beenassumed that the penetration step is a logical consequence of theattachment, producing spontaneous fusion at the surface membrane orpenetration after receptor-mediated endocytosis. In some cases, a secondinteraction has been considered, but this is of a less specific kindthan the attachment, mainly a hydrophobic interaction with the membraneinterior (see reference 2). In the course of the research leading to thepresent invention, however, it has surprisingly been found that, on thecontrary, where certain viruses are concerned, the second binding isspecific, being ascribable to a specific substance with definablechemical characteristics. This binding substance is therefore comparableto the known first-step receptors (see reference 3) which mediateattachment of the virus to the host cell, and is analogously termed thesecond-step receptor. It seems likely that both the first-step and thesecond-step receptors are required on a cell for infection to occur. Theprincipal difference between the two receptors is that the first-stepreceptor is present on the specific cell type which is prone toinfection with a particular species of virus, while the second-stepreceptor is present on all cells which are the ports-of-entry of viralinfections in general (i.e. this receptor is not specific to aparticular viral species). Thus, a virus selects its host cell or tissueby means of the first-step receptor, but uses a generally availablereceptor for penetration.

It is further assumed that the second-step receptor is required forviral survival. Without membrane penetration into the cytoplasm, theattached viral particle would ultimately be degraded in the lysosomesthrough the efficient cleaning machinery of the cell. The second-stepreceptor-binding property is therefore assumed to be a highly conserved(genetically stable so that it does not change, conversely to theantigenic properties of the virus) property of the virus, regardless ofthe nature of the first-step receptor.

A supporting argument in favour of the presence of the second-stepreceptor is the fact that a hydrophobic part of the virus, for instancea hydrophobic peptide sequence such as the peptide sequence constitutingthe binding site on the virus, cannot spontaneously penetrate into thehost cell membrane because of the natural resistance of the membrane.The natural surface membrane is constructed to avoid non-regulateduptake of hydrophobic and other substances. This would otherwiseseriously interfere negatively with the membrane functions. Manyregulatory substances in the organism such as hormones are hydrophobic,and their uptake is mediated by specific receptors and not by meresolubility in the lipid bilayer of the cell membrane. Therefore,analogously, a virus particle in need of hydrophobic penetration mustutilize a bilayer-close receptor which means that an attachment to thefirst-step receptor is not sufficient in itself.

The seemingly paradoxical phenomenon that a virus receptor common tomany cells allows an infection of certain cells only may be explained bythe complex nature of the host cell surface which has a layer ofglycoconjugates extending 100 Å or more from the lipid bilayer. Thesecond-step receptor is located immediately adjacent to the bilayer andis therefore assumed to be hidden from direct contact from the outside.The first-step receptors, on the other hand, are located at a greaterdistance from the bilayer and are therefore immediately accessible forbinding. It is presumed that this first-step binding allows aproximation of the virus particle to the bilayer-close second-stepreceptor which is exposed by the well-known phenomenon of lateralmobility of surface components which is partly induced by repulsion fromthe virus surface due, for instance, to negative electrical charging(see reference 4). The binding of the virus to the second-step receptortherefore requires a first-step attachment which defines the cellspecificity of infection.

The purpose of the present invention is to utilize this basic researchconcerning first-step and second-step binding to employ the naturalsecond-step receptors as such to compete with the second-step receptorspresent on potential host or target cells for the binding sites on thevirus, and by blocking these sites prevent viral penetration into thecells so that viral infection may be prevented or controlled, as well asdevelop compounds which, because they mimic the binding characteristicsof the natural receptor, fulfil the same function of binding to andblocking the binding sites on the virus. It is preferred for the presentpurpose to employ the second-step receptors or similar compounds ratherthan the first-step receptors, as the former are less specific andtherefore have a broader (more general) applicability.

Accordingly, the present invention relates to the use of a compoundcomprising at least one portion, which, with respect to its conformationand properties, corresponds to or is analogous to the binding epitope ofa second-step virus-binding receptor on an animal or plant cell in thatthis portion is capable of binding to a site on a virus which recognizesthe binding epitope of the second-step binding receptor, for themanufacture of a composition for the diagnosis, prophylaxis or treatmentof viral infections.

In the present context, the term "binding epitope" is intended to meanthe smallest possible part of the receptor to interact with the virus.In nature, this binding epitope is carried on the second-step receptorof cells which are targets of viral infections, which receptors aregenerally glycolipids. The existence of the second-step receptorcomprising such a binding epitope has been demonstrated by the presentinventors by means of an assay in which potential receptors inglycolipid form extracted from target cells for infection are separatedon a surface (chromatogram) which by a suitable treatment is made toresemble the biological cell membrane. This means that the potentialreceptor substances are likely to be exposed in the assay in a similarway to the presentation on the living cell. The results from this assaytherefore compare well with binding results from intact cells ortissues. The assay has revealed the presence of first-step andsecond-step receptors, and has furthermore revealed those receptorcandidates which actually do effect binding.

The findings from this assay contradict previously published resultswhich are based on assays in which extracted substances in solubilizedmicellar form are used to inhibit virus attachment to host cells toprovide information on activity. These assays often produce unspecificbinding and therefore do not adequately reflect the actual bindingmechanisms present on the cell surface.

For instance, reference 6 describes the inhibition of hemolysis (leakageof red blood cells) induced by Sendai virus by means of severallong-chain fatty acids, that is, an apparent effect on viruspenetration. Free fatty acids, however, are not components of thesurface membrane. It seems that, in this case, the hydrophobic paraffinchains interact unspecifically with the hydrophobic peptide sequence ofthe F glycoprotein (defined below). Two papers (see reference 7) reportthe inhibition of rhabdovirus by phosphatidylserine and otherphospholipids. Finally, two papers by Huang (see reference 8) showinhibition of hemolysis caused by Sendai virus and Fowl plague virususing phospholipids and some natural and synthetic glycolipids. Thus, itwas possible to inhibit Sendai virus-induced hemolysis using fattyacids, or phospholipids, or glycolipids, presented in micellar form. Asnoted, no binding of Sendai virus by free fatty acids, phospholipids orthe natural or synthetic glycolipids used by Huang was found when usingthe assay developed by the present inventors as described above. Thethree groups of substances are active at comparable levels, but thepolar head groups of the two latter are structurally too different toexplain a specific interaction of the virus with this part. Therefore,the most likely explanation is that the common part of these substances,the lipid chains, interacts hydrophobically in an unspecific way withthe hydrophobic parts of the virus, most likely the N-terminal peptideof the F glycoprotein. This means that the substances used by previousresearchers cannot be genuine receptors on the host cells. In case ofthe phospholipids, this conclusion is supported by the number ofreceptor sites, 10⁶, found on target cells, which is several orders ofmagnitude too low to correspond to phospholipid, a very common cellsurface component.

In brief, the data presented in the literature published to date may beexplained by an unspecific hydrophobic interaction of the testedsubstances with a limited number of viruses. Although this interactionmay theoretically occur with a part of the same peptide inferred belowto bind specifically to the second-step receptor with a structurallydefined binding epitope, there is no evidence presented in thesereferences for genuine receptor specificity. Furthermore, thehydrophobic interaction recognized is common in many systems. Oneexample is fatty acids of different structures associated with albumin,the major protein in blood plasma of mammals. Therefore, the referredfindings cannot be employed for the purposes of the present inventiondue to the generality of simple hydrophobic interactions.

By means of the assay developed by the present inventors, it hastherefore become possible, for the first time, to define specificsubstances which are useful as second-step receptors. Thus, as mentionedabove, in the assay there is an absence of binding to other membranecomponents which have been claimed by other researchers (cf. thereferences indicated above) to bind the viruses analyzed. For instance,there is no binding to free fatty acids/lipids or free carbohydrates,both of which are components of the natural second-step receptor, nor tophospholipids like phosphatidylcholine, sphingomyelin,phosphatidylethanolamine or phosphatidylserine, using relevant levels ofreceptor material. Therefore, the specificity of the assay employed bythe present inventors has made it possible to define the structure ofthe second-step receptor by avoiding the misleading results obtained byother assay techniques, principally the unspecific interaction betweenthe components employed in the traditional assays.

On the basis of the substances found to be receptor-active in the assaydescribed above, certain general characteristics of the binding epitopehave been established. Thus, the compounds used for the purposes of thepresent invention should comprise a hydrophobic part, a polar partadjacent to the hydrophobic part and a part which is both polar andhydrophobic adjacent to the polar part. Furthermore, it has beenestablished that these three parts should form a continuous surface witha total length of about 15-20 Å and a width of about 8-10 Å.

In accordance with the present invention, the hydrophobic part maycomprise a hydrocarbon moiety presenting a hydrophobic surface with asurface area of at least about 50-80 Å². This surface area has beenfound sufficient to establish a hydrophobic interaction between thebinding site on the virus and the hydrophobic part of the bindingepitope, but of course the hydrocarbon moiety may extend over a largerarea which may be practical for some applications where the receptor isto be coupled to a carrier (see below). It is, however, important tonote that a larger surface area of the hydrocarbon moiety does notcontribute to the binding epitope itself. The polar/hydrophobic partshould preferably include a structure corresponding to the α side of thehexose occurring in the natural binding epitope. This α side constitutesthe "hydrophobic" side of the saccharide. The polar part which forms anintermediate zone between the hydrophobic part and the polar/hydrophobicpart should comprise at least two hydrogen-bonding sites. Theintermediate zone may carry both hydrogen bond donors and hydrogen bondacceptors.

The polar/hydrophobic part may comprise a homo- or heterocyclicstructure and is preferably a monosaccharide which is optionallysuitably substituted in such a way that the substituent does notinterfere sterically with the structure corresponding to the α side ofthe hexose occurring in the natural binding epitope. Experiments haveestablished that substitutions that block the binding protrude from thesame side as the α side of this hexose, while substitutions allowing abinding protrude in other directions which indicates a stericspecificity in the approach from the virus. The substituent may, forinstance, be a saccharide. Preferably, the monosaccharide has theconformation of β-galactopyranose or β-glucopyranose, at least in thereceptor-active part thereof, and is preferably a hexose although apentose or heptose may also be employed for this purpose. The hexose maybe galactose or glucose which are the hexoses occurring in the mostoptimal natural second-step receptor.

The hydrophobic part comprised by the hydrocarbon moiety need have nostructural specificity as it does not appear to be essential for primaryepitope recognition and hydrogen bond breaking of membrane resistancewhich is effected by the polar/hydrophobic part and the polar part,respectively. However, the hydrophobic part is important for an extendedhydrophobic interaction between the binding site on the virus and thecell membrane and is therefore still needed in substances used to mimicthe natural receptor as proper binding and therefore blocking of thesite on the virus would otherwise not be effective. The hydrocarbonmoiety may comprise a saturated or unsaturated, branched or linear,open-chain or cyclic hydrocarbon or a combination thereof. Preferably,however, the hydrocarbon moiety comprises one or two saturated orunsaturated, linear or branched hydrocarbons. Most preferably, thehydrocarbon is part of a ceramide as ceramide structures show aself-condensation effect in a monolayer of the surface balance (seereference 33), probably due to intermolecular hydrogen bonding. This maymake the presentation of the binding epitope at a condensed surfaceimportant for efficient binding of a ligand, i.e. the virus. Theceramide should preferably comprise a 2-hydroxy fatty acid as thisappears to improve the accessibility of the virus to the binding epitopein that, in certain of the receptor substances, it provides a suitableconformation and presentation of the binding epitope in a highlyself-condensing monolayer with tight molecular packing due tointermolecular hydrogen bonding. The hydrocarbon moiety should have atleast about 14 carbon atoms, but from a practical point of view (whenproviding sufficient hydrophobicity to bind the hydrocarbon moiety to acarrier by hydrophobic interaction (see below)), preferably about 20-30carbon atoms.

As indicated above, the structure or conformation of the compound to beemployed as the receptor substance or receptor substitute has been foundto be critical for its efficiency in this respect. It has been foundthat the compound should have a bent or curved conformation with apolar/hydrophobic head group bending towards the plane of the monolayerformed by the hydrophobic part such as the hydrocarbon moiety.Accordingly, the compound preferably has a general conformationcorresponding to that of the natural binding epitope carried by1-O-β-D-galactopyranosyl-N-(2-D-hydroxyalkanoyl)-1,3-D-dihydroxy-2-D-aminoalkanewhich has the conformation ##STR1## of the approximate steric atomicrelations described according to crystallographic conventions with acrystal unit cell of a=11.2 Å, b=9.3 Å, c=46.5 Å, and β=99° , and withthe selected fractional atomic coordinates for x, y and z, respectively,for the hexose C1" 0.82, 0.99 and 0.42, C3" 0.91, 1.25 and 0.44, C5"0.72, 1.15 and 0.46, for the long-chain base O1 0.80, 0.90 and 0.41, C10.76, 0.76 and 0.42, C2 0.78, 0.66 and 0.39, C5 0.61, 0.62 and 0.31, N10.90, 0.69 and 0.38, O2 0.57, 0.69 and 0.37, for the fatty acid C1'0.98, 0.58 and 0.37, C2' 1.08, 0.64 and 0.36, C5' 1.17, 0.71 and 0.29O1'0.95, 0.45 and 0.38, O2' 1.12, 0.79 and 0.37,

the compound showing these atomic relations and coordinates or variantsthereof which do not interfere with the binding activity, in that thecompound comprises a polar/hydrophobic part (similar in structure to thepart A shown in the conformation formula of the natural binding epitope)including a structure/conformation corresponding to the α side of thehexose ocurring in the natural binding eptiope and preferably comprisingthe D or L forms of a monosaccharide having the conformation ofβ-galactopyranose, at least in the receptor-active part, whichmonosaccharide is optionally suitably substituted at a position in whichthe substituent does not sterically interfere with the structurecorresponding to the α side of the hexose occurring in the naturalbinding epitope, a polar part (similar in structure to part B shown inthe conformation formula of the natural binding epitope) comprising atleast two hydrogen-bonding sites which may be an amino, carbonyl,hydroxyl, sulfhydryl, sulfoxy or sulfone group, and a hydrophobic part(similar in structure to part C shown in the conformation formula of thenatural binding epitope) comprising a saturated or unsaturated, branchedor linear, open-chain or cyclic hydrocarbon or a combination thereofwith a surface area of at least about 50-80 Å².

From what is outlined above, it appears that the structure of thebinding epitope of the compound employed for the present purposes shouldpreferably be one which closely mimics the conformation of the mostoptimal natural receptor as determined by X-ray crystallography (seereference 31). This natural receptor has a spoon-like or shovel-likeconformation (apparent from the conformation formula shown above) with ahexose (A) bending towards the plane of the monolayer formed by theparaffin chains (C). This means that the α side of the hexose, but notthe β side, is exposed on the outside of the monolayer, as seen from thecrystal packing. The corresponding non-binding glycolipid which differsonly in having a non-hydroxy fatty acid as the hydrophobic part insteadof a 2-hydroxy fatty acid has been studied by NMR spectroscopy (seereference 32) which shows that the hexose is placed perpendicularly tothe plane of the monolayer, and thus rotated around C₁ -C₂ of thelong-chain base. Although the studies were performed by separate methodsapplied to separate molecules, they establish the preferredconformations which differ in a way which is relevant in the presentcontext. Similar to the dense intermolecular packing in the crystal,ceramide structures show a self-condensation effect in a monolayer onwater (and probably also the biological membrane) which is due tointermolecular hydrogen bonding (see reference 33). For the closelypacked glycolipids compared, this means that the non-hydroxy fatty acidspecies presents neither the α nor the β side of the hexose for bindingin contrast to the 2-hydroxy fatty acid-containing species. From this,it may be concluded that the α side of the hexose is involved in thebinding of the virus and that the 2-hydroxy group is critical for makingthis side accessible for binding.

A second factor which is important for receptor epitope designation isthe nature of the protein on the virus which most probably binds thisreceptor. For instance Sendai virus (belonging to the paramyxo virusgroup and used extensively for experimentation) has two proteins on itssurface (see reference 2 and 3). The HN (hemagglutinin-neuraminidase)protein carries the binding site for the first-step receptor containingneuraminic acid. The second protein, the F (fusion) protein, has beenshown to be essential for the fusion of the viral membrane with the hostcell plasma membrane (penetration), which fusion is necessary fordelivery of the viral nucleocapside to the cytoplasm (see reference 2and 3). The involvement of the highly conserved N-terminal hydrophobicpeptide sequence of the F protein in this process has recently beensubstantiated by inhibition with a series of synthetic peptides similarto the natural sequence (see reference 35). Analogous N-terminalpeptides exist on the surface of other viruses (see reference 2, Whiteet al.). The material presented in reference 35 shows that the site ofaction of these synthetic peptides is on the target cell and that theeffect is saturable, indicating receptor sites of about 10⁶ per cell.The second-step receptor as defined herein should be identical withthese sites since there are only two proteins on the Sendai virussurface and two binding properties. The most potent inhibitory syntheticpeptide is the N-carbobenzoxy peptide Z-D-Phe-L-Phe-Gly (Z denotescarbobenzoxy). The N-terminal sequence of the virus surface protein isL-Phe-L-Phe-Gly. The unnatural carbobenzoxy group increases the effectabout 10³ times compared to the peptide without this group. Theunnatural D isomer is 500 times more potent than the corresponding Lisomer.

These two sets of information, the crystal conformation of the receptorglycolipid and the structure of the inhibitory peptides most likelyinteracting with this receptor, have been used for molecular modellingof probable associations of peptide and receptor. Based on the logicalassumption that the N-terminal part of the peptide should be closest tothe bilayer, the optimal fit of the synthetic peptide mentioned aboveand the receptor conformation may be defined as follows. It should benoted that the nomenclature used to denote the position of atoms orfunctional groups in the molecule is according to reference 38.

1. Hydrophobic interaction between the benzoxy group of the peptide andthe paraffin chains of the receptor fatty acid and long-chain base.

2. Hydrogen bonds between N of D-Phe and C═O of the fatty acid and OH3of the base and C═O of D-Phe.

3. Hydrophobic interaction between the benzene ring of D-Phe and the αside of the hexose and CH₂ 1 of the base, including CH1, CH3 and CH5 ofthe hexose ring. It is obvious that a replacement of D-Phe with thenatural L-Phe does not allow such an intimate association.

4. The benzene ring of L-Phe of the synthetic peptide may interacthydrophobically with the paraffin chains of this and adjoined receptormolecules. Thus, a hydrophobic interaction is satisfied for all threebenzene rings.

The curves from quantitative binding assays (see below) indicate alow-affinity binding of the natural receptor to individual sites on thevirus, needing multivalency for efficient binding. This explains why thesynthetic soluble univalent peptide with the natural sequence is of lowpotency for inhibition. The role of the unnatural N-carbobenzoxy groupis to improve the binding by additional hydrophobic interaction with thereceptor compared to the natural structure. This group and the bettersteric fit with the unnatural D-Phe than L-Phe are not needed in case ofa multivalent binding through the natural peptide of the intact virusparticle, providing a sufficiently strong total interaction. This mayindicate that the hydrophobic receptor part interacting with thecarbobenzoxy group is not functional for the virus. However, it isanticipated that an established interaction between virus and a hostcell membrane may include a further hydrophobic area than defined by theepitope as outlined above, after a primary interaction and breaking ofthe membrane resistance has been effected.

Viruses known to carry hydrophoic N-terminal peptides show a slightvariation in the sequence although hydrophobicity is required. Also,there are different effects of inhibition for separate viruses usingvarious synthetic N-carbobenzoxy peptides, with a relative reversal ofpotency in some cases (see reference 35). Extended molecular modellingshows that this "wobbling" in peptide structure is tolerated andproduces a good interaction with the receptor. It should be realizedthat the binding to the receptor, according to this concept, is mediatedby a linear peptide and not by a pocket formed by several peptide loops,often producing both a higher specificity and less tolerance to aminoacid substitutions. On the other hand, a linear peptide is probably moreefficient in gaining access to this partially buried epitope.

Based on the conformation of the natural second-step receptor, thespecifications of a binding epitope carried by the compound to be usedaccording to the invention may be outlined by comparison to similarparts on the natural epitope.

A. The α side of the hexose (Galβ or Glcβ) and C1 of the long-chain base

This is the "hydrophobic" side of the sugar with hydrophobic interactionsites at CH1, CH3, CH4 and CH5 of Gal and CH1, CH3 and CH5 of Glc. Also,CH₂ 1 of the long-chain base represents hydrophobicity. Gal and Glc bindmore or less equally well, showing that the stereochemistry at C4 is notessential. Furthermore, substitution at C4 does not block the bindingbut makes it weaker. These are the only known hexoses of monoglycosylceramides on the mammalian cell. The corresponding part on a syntheticepitope analogue may be comprised of a C₅ -C₇ monosaccharide withsimilar characteristics on the α side of the hexose ring. This may besubstituted in position 4 with a saccharide or another substituent whichdoes not interfere sterically with the access to the binding epitope.Molecular modelling (see reference 38) of probable conformations of anysuch saccharide extensions shows that the substitutions that block thebinding protrude from the same side as the α side of Glc while thesubstitutions allowing a binding protrude in other directions. Thisindicates a steric specificity in the approach from the virus andsupports the statement that the α side is involved in the binding. Inprinciple, the polar/hydrophobic part may in fact be any cyclicstructure which carries the traits of the α side of Gal or Glc, i.e. amainly hydrophobic ring part with extending polar substitutions in theform of, e.g., OH, SH or ═O. Some of these substitutions may benon-polar in the form of, e.g., halogen, CH₃, a shorter alkane oralkene, to optimize the balance between hydrophobic and polar residues.

B. Hydrogen bonding sites at C═O of fatty acid (bond acceptor) and OH3of the base (bond donor and acceptor)

OH4 of phytosphingosine is not essential. OH2 of the fatty acid appearsto be essential for the conformation of the receptor but not for directinteraction with the virus. As noted above, however, an establishedinteraction after the primary specific epitope recognition may also makeuse of this OH for hydrogen bonding. The two bonding sites may be placedabout 5-6 Å apart, and they may primarily be C═O, O═S═O, OH or NH.

C. The hydrophobic part

Based on the molecular modelling, this should include up to C6 of theacid and C8 of the base. However, as noted above, the natural peptidehas only one interaction while the unnatural peptide also bindshydrophobically with the N-carbobenzoxy group, making a precisedefinition uncertain. Again, an established interaction between virusand cell membrane may, after primary epitope recognition and hydrogenbond-breaking of membrane resistance, include an extended hydrophobicinteraction between peptide and membrane. This essential hydrophobicpart may therefore vary and has no structural specificity. In asynthetic epitope analogue, the hydrophobic part may consist of asaturated or unsaturated, branched or linear, open-chain or cyclichydrocarbon, or a combination thereof with a surface area of at leastabout 50-80 Å². A sufficient hydrophobicity may, however, also becreated by the hydrophobic side of a rigid oligomer such as a shortchain of polystyrene, polyethylene, polyvinyl, etc.

The dimensions of the natural epitope which may be concluded from thecrystal conformation (see reference 31) are as follows: the hydrogenbonding sites of part B are situated about 6-8 Å from the centre of thehexose ring of part A. Part C is about 6-8 Å and about 8-10 Å broad. Thetotal length of the binding epitope is therefore on the order of about16-20 Å. A synthetic epitope analogue should therefore have dimensionsof the same or approximately the same size.

As indicated above, the monosaccharide forming the polar/hydrophobicpart of the epitope may be a pentose, hexose or heptose such asnaturally occurring monosaccharides, e.g. xylose, arabinose, glucose,galactose, fucose, ribose, tallose and mannose, or derivatives thereofsuch as deoxy sugars, acetylated or alkylated sugars, branched sugars,amino sugars and uronic acids. In order to possess the desiredcharacteristics with respect to conformation in particular, themonosaccharide should preferably be in ring (such as furanose orpyranose) form. Suitable examples of such a monosaccharide arexylopyranose, arabinopyranose, glucopyranose, galactopyranose,mannopyranose, etc. As further indicated above, this monosaccharide maybe substituted at a position where the substituent does not interferesterically with the binding. For most monosaccharides, this means thatthe substitution may occur at position 4 (carbon atom 4 of theheterocyclic ring). In principle, the substituent may be any substituentwhich fulfils the above criterion and which furthermore does not disturbthe balance between polarity and hydrophobicity in this part of theepitope. Preferred substituents are mono-, di-, tri-or tetrasaccharidessuch as Gal, Glc, Xyl, Ara, Fuc, Rib, Man, Man-α1→3Man, Manα1→2Man,Manα1→6Man, Galβ1→2Man, Fucα1→2Gal, Fucα1→3-Gal, Fucα1→6Gal, Galβ1→2Gal,Galβ1→6Gal, Galβ1→3Gal, Galα1→3Galβ, Galα1→4Galβ, Glcα1→4Glc,Glcβ1→4Glc, Glcα1→4Glcβ1→4Glc, Glcα1→4Glcα-1→4Glc, Galβ1→3Galβ1→4Glc,Galβ1→3Galβ1→3Galβ1→4Glc, Glcα1→6Glcα1→4-Glcα1→4Glc,Galα1→3(Fucα1→2)Galβ, any suitable combination thereof or a derivativethereof such as a suitably acetylated, alkylated, branched or aminatedderivative thereof (the abbreviations used are in accordance withaccepted carbohydrate nomenclature).

The three parts constituting a synthetic binding epitope analogous tothe natural binding epitope should be joined to present a total bindingepitope of the indicated dimensions and with the characteristicsdescribed above exposed on the same side of the designed surface. Thismay be achieved by synthesis of a rather rigid epitope similar to thenatural epitope. Thus, the link between the polar/hydrophobic part andthe polar part may comprise two suitably substituted carbon atoms withthe conformation of the natural binding epitope. Furthermore, the linkbetween the polar/hydrophobic part and the polar part may be establishedby a linkage between glycosidic oxygen or glycosidic sulphur or an etheror thioether on the polar/hydrophobic part and a CH₂ group, or a similargroup, on the polar part. If the coupling between the polar/hydrophobicpart and the polar part is to be short and rather rigid, the glycosidiclinkage is of β anomerity. However, the three parts may also be joinedmore flexibly to each other provided that the nature and length of thelink does not interfere with the adoption of the binding surface of thethree parts with the dimensions outlined above. Provided this criterionis satisfied, the specifications of A in particular may be more varied;if used, the anomeric linkage may for instance be of the α type.

As indicated above, receptors on target cells are most often eitherglycoproteins or glycolipids. As appears from the conformationspecifications for the natural receptor, however, it has been foundthat, in nature, only glycolipids may function as second-step receptorsfor virus binding. The present invention is therefore primarilyconcerned with the use as antiviral agents of lipid-linkedcarbohydrates, including glycolipids, glycosphingolipids andglycoglycerolipids, or receptor-active analogues thereof. Examples ofglycolipids useful for this purpose are D-galactopyranosyl-β-diglycerideor D-galactopyranosyl-α1→6-D-galactopyranosyl-β-diglyceride, which, innature, may be found on the surface of plant cell membranes and havebeen found to possess the properties of the second-step bindingreceptor, indicating their usefulness in combating plant diseases ofviral origin.

The intermediate zone of glycoglycerolipids shows hydrogen bondacceptors only and therefore does not cause the tight molecular packingand highly self-condensing monolayer which have been indicated to beimportant causes of the proper conformation and presentation of thebinding epitope in the case of glycosphingolipids. Experiments haveshown that the binding to glycoglycerolipids does not require a2-hydroxy fatty acid, as the binding epitope is available to the viruseven in the absence of 2-hydroxy fatty acid due to the more spacedpresentation of the glycoglycerolipid molecules. Whereglycosphingolipids are concerned, it is preferred that the hydrophobicpart comprises a 2-hydroxy fatty acid as this has been shown to cause amore efficient binding. Accordingly, the compound to be used as anantiviral agent may be a sphingosine-2-D-hydroxy fatty acid of thegeneral formula I or a phytosphingosine-2-D-hydroxy fatty acid of thegeneral formula II or a dihydrosphingosine-2-D-hydroxy fatty acid of thegeneral formula III ##STR2## wherein R represents a saccharide with theconformation of β-galactopyranose, at least in the receptor-active part,and R₁ and R₂ each independently represent a methyl group, or a CHO,NO₂, NH₂, OH, SH, CONHNH₂, CON₃ or COOH group. When the receptor orreceptor analogue is to be used as such, i.e. is efficient enough toeffect binding in itself, R₁ and R₂ are preferably methyl, but when thenatural epitope or the synthetic epitope analogue are to be presented ina multivalent form (see below), it is preferred that R₁ and/or R₂represents a reactive group which is capable of either interacting withsimilar groups on the receptors themselves or reacting with similargroups on a carrier. R₃ represents a hydrocarbon moiety which maycomprise a linear or branched, saturated or unsaturated hydrocarbon witha chain length of at least 5 carbon atoms. However, the hydrocarbonchains are most usually linear and saturated or monounsaturated. R maybe a monosaccharide as specified above, optionally substituted inposition 4 by one or more other saccharides as mentioned above; thus,the total saccharide comprising the polar/hydrophobic part may forinstance be

Galβ,

Glcβ,

Galβ1→4Glcβ,

Galα1→4Galβ,

Galα1→3Galβ1→4Glcβ,

Galα1→4Galβ1→4Glcβ,

Fucα1→2Galβ1→4Glcβ,

GlcNAcβ1→3Galβ1→4Glcβ,

GalNAcβ1→4Galβ1→4Glcβ,

Galα1→3(Fucα1→2)Galβ1→4Glcβ,

Galβ1→3GlcNAcβ1→3Galβ1→4Glcβ,

Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ,

Galβ1→3(Fucα1→4)GlcNAcβ1→3Galβ1.fwdarw.4Glcβ, or

Galβ1→4(Fucα1→3)GlcNAcβ1→3Galβ1.fwdarw.4Glcβ,

wherein the term "NAc" denotes an N-acetylation of the saccharide.

It has been experimentally established that glycolipids with up to fivesaccharides are able to bind the viruses; a larger oligosaccharide islikely to constitute a steric hindrance in the access of the virus tothe epitope. The best natural binders, as established by quantitativebinding studies (see below), have been found to be GlcβCer and GalβCer.This indicates that the use of more than one saccharide is superfluousfor binding purposes although it may be necessary from the point of viewof the availability of starting materials in the preparation of thereceptors or receptor analogues, etc.

As repeatedly indicated above, the use according to the invention alsoextends to receptor-active analogues of the natural binding epitope.Such analogues may, of course, have any form provided that they meet therequirements with respect to conformation described above, and suchanalogues may therefore be selected from a wide variety of compounds. Ithas, however, been found that compounds of the general formula IV##STR3## wherein R, R₁, R₂ and R₃ are as defined above, form aparticularly interesting group of receptor analogues to be employed forthe present purpose. In the case of the synthetic receptor analogues,the same saccharides as those listed above for natural receptorsubstances may be employed. Particularly interesting compounds of thisgeneral formula are ##STR4## which have been found to show an avidity ofbinding which is on the same order of magnitude as that of the naturalreceptor substances (cf. Example 5).

As these receptor analogues have been found to be comparable to thenatural substances with respect to properties and conformation(dimensions), they are fully valid as substitutes for the naturalreceptor. The advantage of using these or similar synthetic receptoranalogues lies in the method of their preparation which may be effectedby simpler means than if the natural receptor were to be produced eithersynthetically or by the more cumbersome method of harvesting them fromcell surface membranes.

The synthetic receptor analogues of the general formula IV may beprepared by a process comprising reacting a glycoside of the generalformula V ##STR5## wherein X represents a leaving group and R is asdefined above, with a thiol of the general formula VI

    HS--R.sub.3 --R.sub.1                                      VI

wherein R₃ and R₁ are as defined above and reacting the product with anoxidising agent. The reaction may be carried out in water or a suitableorganic solvent such as ethyl acetate, methylene chloride, ether anddimethyl sulfoxide. The reaction may suitably be carried out at roomtemperature. The reaction time may be from 24 to 48 hours. The reactionis generally carried out with slightly more than 2 equivalents of thethiol of the general formula VI to one equivalent of the glycoside ofthe general formula V. The reaction may be carried out under slightlyalkaline conditions, and the saccharide may be suitably protected.

The oxidation of the resulting thio compounds to the correspondingsulphones of the general formula IV may take place using fourequivalents or more of the oxidizing agent. Examples of useful oxidizingagents are peracids such as m-chloroperbenzoic acid, peroxides such astert.butyl hydroperoxide, aminooxides, gaseous oxygen or inorganicoxidizing agents such as potassium permanganate, chromium trioxide, etc.The reaction is usually carried out at room temperature.

Another interesting group of compounds are compounds of the generalformula VII

    ROCH.sub.2 CH.sub.2 SO.sub.2 --R.sub.3 --R.sub.1           VII

wherein R, R₁ and R₃ are as defined above. These compounds may beprepared from the corresponding sulfides by oxidation as describedabove.

In order to be useful as a diagnostic, prophylactic or therapeutic agentin connection with viral infections, it is important that the compoundused for these purposes is provided in such a form or in such a way thata sufficient affinity for the virus is obtained. Thus, the compound, ifsufficiently active, may be provided as such in water-soluble form, i.e.in univalent form (that is, appearing as discrete units of the compoundeach carrying substantially only one binding epitope). This form isexpected to be particularly relevant where synthetic receptors areconcerned as these may have a far greater affinity for the viruses thanthe natural receptors which have been found to show a low-affinitybinding. Where the natural and, supposedly, several of the syntheticreceptors are concerned, the compound may therefore usually preferablybe provided in such a form that it presents multiple binding sites forthe virus (in the following termed "multivalent binding") in order toproduce an efficient binding. In nature, multivalent binding is effectedby a large number of protein molecules on the virus surface and acorresponding number of glycolipid receptors on the host cell. When usedfor the present purpose, the compound should therefore be provided in aform that mimics this multivalent binding. This may be achieved by theuse of the natural glycolipid receptors or synthetic receptor analoguesof a similar size which may be presented as micelles or on hydrophobicsurfaces (to resemble the target cell surface). However, the less stablemicelles may give rise to unspecific binding to any surface, andtherefore the presentation of the receptor or receptor analogue on asolid phase is preferred.

Alternatively, the compound may be multivalently coupled to amacromolecular carrier which may be either of two types: one in whichthe carrier has a hydrophobic surface to which a hydrophobic part of thecompound is associated by hydrophobic non-covalent interaction, thehydrophobic surface for instance being a polymer such as a plastic orany other polymer to which hydrophobic groups have been linked, such aspolystyrene, polyethylene, polyvinyl, etc., or it may be amacromolecular carrier to which the compound is covalently bound. Inthis latter instance, a suitable carrier may be a natural or syntheticpolymer. Thus, the carrier may be an oligo- or polysaccharide, e.g.,cellulose, starch, glycogen, chitosane or aminated sepharose, to whichthe receptor or receptor analogue may be bonded through a reactive groupon the hydrophobic part, such as a hydroxy or amino group present on thereceptor-active substance, an oligo- or polypeptide such as a globulin,albumin, fibrin, etc., the bond being provided through, for instance, ahydroxy or amino group on the hydrophobic part of the receptor orreceptor analogue, a combination thereof or a similar suitablysubstituted conjugate. The carrier may also be an inorganic carrier suchas a silicon oxide material, e.g. silica gel, zeolite, diatomaceousearth, or the surface of various glass types such as aminated glass towhich the receptor or receptor analogue may be bonded through a hydroxy,carboxy or amino group on the hydrocarbon moiety of the receptor-activesubstance. When the compound is to be used for prophylaxis or therapy,it is vital that the carrier is a physiologically and pharmaceuticallyacceptable carrier, such as a non-toxic and/or non-allergenic carrier.Carriers of this type which are at present contemplated to be useful forthis purpose are, for instance, poly-L-lysine and poly-D,L-alanine.

The present invention is based on the finding that viruses in generalappear to need the second-step receptor for the essential penetrationinto the host cell cytoplasm so that the natural binding epitope or asynthetic epitope analogue in uni- or multivalent form may be employedto prevent viral infection by blocking the available binding sites onthe virus so as to inhibit invasion of the virus into epithelial cellsat the port-of-entry of viral infections. Examples of suchports-of-entry are the mucous membranes of the eye, nose, oral cavity,throat, respiratory tract, gastrointestinal tract, urinary tract andreproductive organs. The general applicability of the present inventionis based on the fact that compounds corresponding to or analogous to thesecond-step binding receptor found on target cells have been shown tobind a wide variety of viruses belonging to the families Adenoviridae,Herpetoviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae,Reoviridae. Other virus families of interest in this connection are thePicornaviridae and Retroviridae. Thus, the viruses of interest includeboth DNA and RNA viruses and particles with and without a bilayermembrane envelope, which viruses are the cause of a wide range ofdiseases affecting many different organ systems, examples of which arevarious influenzas and common colds, diarrhoeas, herpes I and II, mumps,measles, rabies, AIDS, leukaemia, etc.

Prophylaxis in the animal, including human, system may be obtained bydirect application on the mucous membrane of interest of thevirus-inhibiting compound in a pharmaceutically acceptable form such asa suspension, aerosol, ointment, suppository, spray, lotion or solutioncontaining the receptor or receptor analogue in univalent or multivalentform as described above. There, the active substance binds the virus andblocks the binding site for the second-step receptor. Prophylaxis mayalso be effected by exposing the virus particles secreted from infectedpatients or animals to a compound of the invention applied on thesurface of apparatus, objects or surfaces which may be or have beenbrought in contact with such secretions, i.e. the compounds may be usedas disinfectants. Exposure of the virus to a multivalent receptor maynot only prevent the viruses from penetrating into the target cell, butmay also agglutinate the virus particles through multivalentattachments. This means that most virus particles will also be preventedfrom attachment to the first-step receptor. Prophylaxis in the plantsystem may for instance be effected by spraying seeds, seedlings oradult plants with an effective dosage of a receptor or receptor analogueformulated in a manner to facilitate application such as a solution,suspension or emulsion of a receptor or receptor analogue in multivalentor univalent form as described above.

The use according to the invention may also comprise a therapeutic usein cases where infection has already taken place, i.e. when the virushas already passed the mucous membrane at the port-of-entry and settledwithin the organism. Therapeutic administration of the receptor is atpresent contemplated to be achievable by injection or through intestinalabsorption or direct absorption from other mucous membranes of a solublelow-molecular weight univalent receptor analogue with increased virusbinding potency as outlined above. Thus, the injected or absorbedsubstance is expected to bind virus particles that are budding (beingexpelled) after multiplication in infected cells and prevent them fromentering new cells (spread of infection). A similar prevention of spreadis also useful in, for instance, the small intestine after virusinfection by oral presentation on a multivalent carrier, e.g. as tabletsor capsules in acid resistant form. As an example of application afterabsorption may be mentioned rabies infection where, after a bite(wound), the virus travels through the nerve pathways to finally reachthe central nervous system (after a period of several months or evenyears) to produce the lethal effect. A compound as described above madeto penetrate the blood-brain barrier or injected directly into thecerebro-spinal fluid is expected to prevent the brain damage caused byviral multiplication within nerve cells.

The dosages in which the receptors or receptor analogues areadministered may vary widely depending on the intended use, whether forprophylaxis, including disinfection, or therapy, the type of infectionto be combated, the age and condition of the patient, etc., but isexpected to be at a milligram level. For rotavirus infection(diarrhoea), a daily dose of 1 μg receptor per human individual has beencalculated to agglutinate/inactivate all viruses produced during oneday, provided the receptor is bivalent and only one bivalent receptor isused per virus particle. In practice, of course, a far larger dosage isneeded to secure an effective binding of all the virus particlespresent. Contrary to what is the case with most medicaments now in use,the dosage level may not be so essential as the toxic effects of thereceptor or receptor analogues are expected to be negligible since, infact, at least the natural receptors are substances which are present inlarge amounts in the human or animal system.

The use according to the invention may furthermore be a diagnostic use,utilizing the knowledge of the second-step receptor function withrespect to the binding of viruses. For diagnostic purposes, virusparticles present in secretions from infected patients or other samplesto be tested may be exposed to the receptor substance which may bepresented on the surface of an appropriate carrier material in the formof, for instance, dip-sticks, immunological test cards, polymeric beadsor the like. After binding, the surface is carefully washed, and thepresence of the virus may be shown in several ways already establishedwithin clinical virology. For instance, the virus may first be adsorbedin microtiter wells followed by detection by ELISA methods (seereference 1, Materials and Methods and Example 6). Also, the otherassays developed by the present inventors may be adapted for asimplified diagnostic use.

It is also contemplated that the compounds described above may be usedfor various biotechnological purposes such as the preparation andisolation of virus particles or virus surface components through the useof established affinity chromatographic methods. For this purpose, thereceptor substance may be coupled to a solid support such asmultivalently coupled to a macromolecular carrier as described above forbinding the ligand in chromatographic columns, for electrophoresis,filtration, sedimentation or centrifugation. The properties of thereceptor (binding constant, etc.) may be optimized for elution, etc., bythe design of the synthesis of the compound so as to avoid irreversiblebinding of the virus to the receptor. In this way, it may be possible toisolate or purify virus particles or virus substances and/or detectviruses in various samples by their binding to receptor or receptoranalogues coupled to these solid supports.

Another suggested use of the receptors or receptor analogues may be toisolate the substance on the virus membrane which is responsible forvirus binding. It is contemplated that this substance may be used forvaccination purposes, i.e. to immunize the human being or animal inquestion not only against the viral disease imparted by the viralspecies from which the substance has been derived, but also againstother viral diseases imparted by viruses having the same substance ontheir surface. Thus, it is at present assumed that a broad-specteredvaccine against a variety of viral diseases may be produced in thismanner. Compared to the usual methods of vaccination using attenuatedvirus preparations, such a vaccine would have the decided advantage thatnon-infectous virus components may stimulate the production ofantibodies which are able to neutralize the virus in question byblocking the essential molecule for virus entry into the host cell forinfection.

Finally, it is contemplated that it may be possible to select bindingproteins or glycoproteins from the viruses in the manner described abovewhich may be used for targeting drugs to specific cells within anorganism. For instance, a liposome (lipid vesicle) may be equipped with,e.g., a monoclonal antibody with specificity for a certain target cell(for instance a tumor cell or a cell carrying intracellular parasites).To avoid being eventually inactivated through endocytotic uptake to thelysosomes, the liposome may also be tagged with the viral componentrecognizing the second-step receptor. In this way, the established virusmechanism for penetration may be employed as part of a vehicle forcarrying a toxic drug. The liposome may be exchanged with a directchemical coupling of antibody (first-step) and viral component(second-step) with the active subunit of a bacterial toxin exerting itsactions only inside the cell (see reference 12).

The present invention also relates to an antiviral agent which is acompound with the characteristics and properties described above. It isto be understood that this aspect relates to the compound in isolatedform, that is, it does not include the compound when carried by thecells which, in nature, carry the compound.

The present invention further relates to a pharmaceutical compositionwhich comprises a compound with the characteristics and propertiesdescribed above which has been formulated together with apharmaceutically acceptable vehicle or excipient.

The composition may be formulated for administration by any suitableroute such as by oral ingestion, injection or topical application. Thus,the composition may be in the form of, for instance, a suspension,solution, aerosol, ointment, lotion, cream, spray, suppository, implant,tablet, capsule or lozenge containing the receptor or receptor analoguein univalent or multivalent form as explained above. It is contemplatedthat for prophylactic use, the composition may be in a form suitable fortopical application on the mucous membrane of, e.g., the eye, nose, oralcavity and throat, such as an aerosol, spray, lozenge, lotion or cream,and for the purposes of disinfection, the receptor or receptor analoguein question may be provided as, e.g., paper tissues or a liquid which issuited for rinsing apparatus, objects or surfaces which may be or hasbeen brought into contact with secretions from infected patients.

For therapeutic applications, it is contemplated that compositionsadapted for injection, such as solutions or suspensions of the receptoror receptor analogue in a suitable vehicle such as water or isotonicsaline, as well as adapted for oral ingestion (and absorption throughthe intestinal mucosa) such as tablets or capsules will be convenient.In order to protect the receptor or receptor analogue from possibleimpairment or degradation in gastric juices, the tablets or capsules maybe provided with an acid-resistant coating. Compositions adapted forabsorption of the receptor substance through mucous membranes may alsobe in the form of suppositories such as vaginal or rectal suppositories.

The pharmaceutically acceptable vehicles or excipients and optionallyother pharmaceutically acceptable materials present in the compositionsuch as diluents, binders, colorants, flavouring agents, preservativesand disintegrants are all selected in accordance with conventionalpharmaceutical practice in a manner understood by persons skilled in theart of formulating pharmaceuticals.

DESCRIPTION OF THE DRAWING

The invention is further described with reference to the drawing inwhich

FIG. 1 shows an assay of Sendai virus (S variant) binding to thefirst-step receptor, analyzed as described in Materials and Methods. Thetwo lanes to the left have been detected chemically and are totalgangliosides (neuraminic acid-containing glycolipids) of humanerythrocytes (lane 1) and human brain (lane 2). The same two lanes areshown to the right after binding of Sendai virus and autoradiography. Asshown, there is no virus binding to the major brain bands of lane 2. Incontrast, there is an apparently strong binding to several erythrocytebands of lane 1, especially slow-moving ones.

FIG. 2 shows an assay of Sendai virus (S variant) binding to thesecond-step receptor, analyzed as described in Materials and Methods.This illustrates the possible analysis of a large number of receptorcandidates as mixtures of glycolipids of various origins (screening forreceptors) possible with this assay. To the left is the chromatogramafter chemical detection with anisaldehyde and to the right theautoradiogram after an overlay of virus over the same lanes. Thefollowing are the total non-acid glycolipids shown: human erythrocytes(lane 1), human meconium (lane 2), intestine from Macaca cynomolgus(lane 3), dog small intestine (lane 4), rabbit small intestine (lane 5),guinea-pig small intestine (lane 6), and mouse large intestine (lane 7).The white numbers refer to substances listed in Table 3. It appears thata large number of major glycolipid bands detected chemically (left) donot bind the virus (right), indicating the high selectivity andspecificity of the assay.

FIG. 3 shows an assay of Sendai virus (S variant) binding to thesecond-step receptor substances, analyzed as described in Materials andMethods. This illustrates the binding to substances which are syntheticor have been isolated from natural sources and identified chemically. Tosave space, several pure substances were added to the same lane,starting with the most rapid-moving substance as shown in thechromatogram to the left, detected by anisaldehyde. The autoradiogram isshown to the right. The figures refer to Table 3. Lane 1: synthetic 2with phytosphingosine and 2D-hydroxystearic acid, 3 with non-hydroxyfatty acid, 15, andGalNAcα1→3(Fucα1→2)Galβ1→4-(Fuc.alpha.1→3)GlcNAcβ1→3Galβ1→4GlcβCer; lane2: synthetic 2 with phytosphingosine and stearic acid, 3 (three closelymoving bands with 2-hydroxy fatty acid), and 15 with non-hydroxy fattyacid; lane 3: synthetic 2 with sphingosine and stearic acid, 3, 6 withnon-hydroxy fatty acid (contaminated), 16, and 22 (three closely movingbands with 2-hydroxy fatty acid); lane 4: synthetic 1 withphytosphingosine and 2-hydroxy stearic acid, 3 with phytosphingosine andnon-hydroxy fatty acid, 6, and GalNAcα1→3GalNAcβ1→3Galα1→4Galβ1→4GlcβCerwith non-hydroxy fatty acid; lane 5: synthetic 1 with sphingosine andstearic acid, substance 13 of Table 1, 6 (short-chain 2-hydroxy fattyacid), 16 (two closely moving bands with 2-hydroxy fatty acid), and 17(three closely moving bands in the autoradiogram to the right may becontaminants in 17); lane 6: 8 (two closely moving bands), 20, andGalNAcα1→3(Fucα1→2)Galβ1→4GlcNAc.beta.1→3Galβ1→4GlcβCer.

FIG. 4 shows the curves obtained from the quantitative binding (cf.Materials and Methods) of Sendai virus to various synthetic or naturalglycolipids, obtained as described in Materials and Methods. Dilutionsof glycolipids were adsorbed in microtiter wells (x axis) and virusbinding was, after incubation, estimated as radioactivity (y axis). Theletters refer to the following samples tested: A is substance 1 or 2 inTable 3. B is the first-step receptor with the following sequence:NeuAcα2→3Galβ1→4GlcNAcβ1→3(NeuAc.alpha.2→3Galβ1→4GlcNAcβ1→6)Galβ1→4Glc-NAcβ1→3Galβ1→4GlcβCer.C is substance 3 in Table 3 (with 2-hydroxy fatty acid) or substance 18in Table 1. D is substance 5 in Table 3. E may be substances 21 and 22in Table 3 (which bind weakly in the chromatogram, see FIG. 3, lane 3for substance 22) or the substances which are negative in Table 3.

FIG. 5 shows examples of naturally occurring molecular species ofceramides (combinations of fatty acid and long-chain base). These havebeen summarized elsewhere (Karlsson, in Chapman, ed., BiologicalMembranes, Vol. 4, Academic Press, London, 1982, pp. 1-74). R includesthe oxygen of C1 of the base and may be phosphorylcholine as insphingomyelin or a saccharide as in glycosphingolipids. The major typesof fatty acids are non-hydroxy, 2-D-hydroxy and 2-D,3-D-dihydroxy fattyacids with about 12-26 carbon atoms in the chain, which may besaturated, unsaturated, linear or branched. The major types of bases aredihydroxy (sphingosine, dihydrosphingosine and related bases) andtrihydroxy bases (phytosphingosine and related bases). They have about14-22 carbon atoms and the chain may be saturated, unsaturated, linearor branched. The absolute structures of the three classical bases are:Sphingosine: 1,3-D-dihydroxy-2-D-amino-4-trans-octadecene (of species Band D), dihydrosphingosine: 1,3-D-dihydroxy-2-D-aminooctadecane (ofspecies A) and phytosphingosine:1,3-D,4-D-trihydroxy-2-D-aminooctadecane (of species C, E and F). Themost common species of mammalian cells are A-E, often with a 15-cisdouble bond in the fatty acid. The most common species of epithelialcells are D and E, which are the best virus binders (Table 4).Therefore, the viruses have selected, as second-step receptors,glycolipids with a ceramide composition which dominates in theepithelial cells, the port-of-entry of viral infections.

FIG. 6 shows the conformation of the second-step receptor as obtained bysingle crystal X-ray crystallography of synthetic substance 1 of Table 3with dihydrosphingosine and 2D-hydroxy stearic acid (cf. Pascher et al.,Chem. Phys. Lipids 20, 1977, pp. 175-191). The nitrogen is dotted andthe carbonyl oxygen and C20 of the acid are hatched. The substance wassynthesized as described in Pascher, Chem. Phys. Lipids 12, 1974, pp.303-315, and crystallized from 95% of ethanol. The X-ray data werecollected on a Picker FACF I defractometer and more than 5000independent reflections were measured. All calculations were performedon a DEC-10 computer system using mainly the X-RAY 72 programme system.

FIG. 7 shows a thin-layer chromatogram detected with anisaldehyde toillustrate the existence of second-step receptor glycolipids inepithelial cells of the human small intestine. Epithelial cells andtotal non-acid glycolipids were prepared and identified as described inMaterials and Methods. The double bands which are common to the fourlanes have been identified as a mixture of 1 and 2 of Table 3, the twomost optimal natural virus binders (Example 2 and FIG. 4). The moreslow-moving glycolipids are blood group fucolipids and vary among thefour individuals analyzed which were of the following blood groupphenotypes. Lane 1: A₁ Le (a-b+), secretor; lane 2: OLe (a-b+),secretor; lane 3: OLe (a+b-), non-secretor, lane 4: OLe (a-b-),secretor. The composition of these samples has been described by Bjorket al., in Cartron et al., eds., Red Cel Membrane Glycoconjugates andRelated Genetic Markers, Libraire Arnette, Paris, 1983, pp. 125-137.

FIG. 8 shows the structural concept of the outer monolayer of the animalcell surface membrane as described in detail elsewhere (Karlsson, inChapman, ed., Biological Membranes, Vol. 4, Academic Press, London,1982, pp. 1-74). The three major lipid components are sphingolipid,glycerolipid and cholesterol, and in the intermediate zone they carry anumber of hydrogen bond acceptors and donors which create a system ofintermolecular laterally oriented hydrogen bonds of importance formembrane stability. Binding sites are especially dense in epithelialcells of mucous membranes, the port-of-entry of viral infections. Asshown in Example 4, epithelial cells are abundant in the second-stepreceptor (substance 1 and 2 of Table 3), which is a sphingolipidcontaining both hydrogen bond acceptors and donors. Plant cells alsocontain substance 2 of Table 3 in addition to substances 26 and 27 ofTable 3, which are glycerolipids. These bind the viruses (Table 3).Viruses have therefore developed a property (through a surface peptide)which is able to utilize, for selective binding, all three parts of thesurface membrane that are essential to make the membrane a stablebarrier. In this way, the second-step receptor for virus penetration andinfection is a normal component of all cell surfaces.

MATERIALS AND METHODS Preparation and Structural Characterization of theNatural Receptor and Related Substances

The human and animal glycolipid samples used for binding studies, eitherpure glycolipid species or total or partial glycolipid mixtures, wereprepared substantially as described in reference 13 from lyophilizedtissue through repeated chloroform-methanol extractions, mild alkalinetreatment to degrade non-sphingolipids and dialysis for 4 days againstwater to remove water-soluble products. Remaining lipid material isfirst separated on a silica gel column to elute the dominating fattyacid esters before the totally alkali-stable sphingolipids. These arethen separated on DEAE cellulose or DEAE Sepharose® columns in anon-acid and acid glycolipid fraction. The acid fraction is then usuallyseparated by gradient elution from DEAE Sepharose®. The non-acidfraction is acetylated in acetic anhydride and pyridine and repeatedlyfractionated on silica gel with stepwise or continuous gradient elutionwith methanol in chloroform or other solvents. Fractions aredeacetylated in mild alkali and further fractionated until pure ifnecessary. Purity is tested by thin-layer chromatography and furtherchecked by mass spectrometry and NMR spectroscopy as described below.

To prepare glycolipids completely free from non-glycolipid contaminants,250 ml of methanol are added to about 250 ml of human blood plasma ofone transfusion unit, and the mixture is heated to 70° C. for 30 minuteswith constant stirring in a one-liter evaporation bottle. The extract isfiltered and the residue transferred back to the extraction bottle. Theprocedure is repeated twice with 250 ml of chloroform/methanol 2:1 (byvolume) and once with 250 ml of methanol. The combined extracts areevaporated to dryness with the addition of small volumes of toluene.

Small amounts of wet cells may be extracted in a similar way. On alarger scale, the tissue is first lyophilized in pieces and subjected toextraction in two steps in a Soxhlet apparatus. In the case of humansmall intestine (e.g. 130 g dry weight), the first extraction is withchloroform/methanol 2:1 (by volume) for 24 hours (1000 ml of solvent ina 2000 ml round bottle placed in an asbestos-insulated electricalheating device). The second extraction is carried out with 1500 ml ofchloroform/methanol 1:9 (by volume) for 24 hours. The combined extractsare evaporated to dryness without filtration.

The dried plasma extract residue is treated with 50 ml of 0.2M KOH inmethanol for 3 hours in a bottle containing five glass beads for finedispersion during occasional shaking. The KOH is neutralized with 1 mlof acetic acid. The mixture is dialyzed against water in a dialysis bagafter the addition of 100 ml of chloroform and 40 ml of water to producea two-phase system. After dialysis for 4 days against running tap water,the content of the bag is evaporated at 70° C. with repeated additionsof toluene. The sample is finally filtrated and eluted withchloroform/methanol 2:1 (by volume) and methanol. In case of theintestinal total extract, 500 ml of KOH in methanol are used.

The plasma sample (about 1.4 g) is loaded on a 10 g column of silica gelpacked in chloroform. The silica gel used is principally obtained fromMallinckrodt Chem. Works, St. Louis, U.S.A., sieved to a particle sizeof more than 45 μm and dried. However, LiChroprep Si 60 (E. Merck,Darmstadt, West Germany) with comparable specifications has similarproperties although some of the silica gel may elute. Three fractionsare eluted: 100 ml of chloroform elute the main bulk (about 1 g of theextract (cholesterol and methyl esters of fatty acids); 100 ml ofchloroform/methanol 98:2 (by volume) may contain free ceramide; 100 mlof chloroform/methanol 1:3 (by volume) and 100 ml of methanol elute allglycolipids and alkali-stable phospholipids (about 60 mg). In case ofthe dialyzed small intestinal sample (about 40 g), 50 g of silica geland 500 ml of solvent are used in each step.

The third fraction from the silica gel chromatography of the plasmasample is loaded on a column of 5 g of DEAE cellulose (DE-23, Whatman)in acetate form packed in chloroform/methanol 2:1 (by volume). Theloaded sample is allowed to equilibrate on the column for 1 or 2 days.Two fractions are eluted, one with 100 ml of chloroform/methanol 2:1 (byvolume) and 100 ml of methanol eluting non-acid glycolipids andalkali-stable phospholipids, mainly sphingomyelin, and one with 50 ml of5% (w/v) LiCl in methanol eluting acid glycolipids (sulphatides andgangliosides) and alkali-stable phospholipids. The latter fraction isdialyzed with 30 ml of chloroform and 20 ml of water against running tapwater for 4 days. It may be used as a total acid glycolipid fraction orbe further processed as described to separate sulphatides andgangliosides (Breimer et al., J. Biochem. 93, 1983, pp. 1473-1485). Thefirst fraction is evaporated to dryness and acetylated. In case of theintestinal sample (about 2 g), 20 g of DEAE cellulose are used.

Acetylation of the dry non-acid plasma sample is performed in the darkovernight in 2 ml of chloroform, 2 ml of pyridine and 2 ml of aceticanhydride. The chloroform is added to improve solubility and ensurecomplete reaction. 5 ml of methanol and 5 ml of toluene are added, andthe sample is evaporated in a stream of nitrogen on a heated water bathand finally subjected to vacuum suction.

The acetylated plasma sample is loaded on a 10 g column of silica gelpacked in chloroform/methanol 98:2 (by volume). For this purpose,particles smaller than 45 μm treated with methanol and dried are used.Three fractions are eluted: 100 ml of chloroform/methanol 95:5 (byvolume), 100 ml of chloroform/methanol 90:10 (by volume), and 100 ml ofchloroform/methanol 1:3 (by volume) plus 100 ml of methanol. The thirdfraction contains mainly acetylated sphingomyelin. The first twofractions which contain acetylated glycolipids and some contaminants areevaporated together and deacetylated. For the intestinal fraction, 50 gof silica gel are used.

Deacetylation of acetylated glycolipids may be performed in two ways,one with and one without a dialysis step.

Method A. For the plasma sample, 2 ml of toluene, 2 ml of methanol and 4ml of 0.2M KOH in methanol are used with occasional shaking during 30minutes (for the intestinal sample, 5, 5 and 10 ml, respectively, areused). After addition of 0.5 ml of acetic acid, the sample istransferred with 10 ml of chloroform and 10 ml of water to a dialysisbag (two-phase system) and dialyzed for 4 days against running tapwater. The content of the bag is evaporated at 70° C. with repeatedadditions of toluene.

Method B. In this case, no dialysis is needed since the amount ofpotassium acetate formed after neutralization is very low compared tothe glycolipid. The reagent is composed of 1 ml of 0.2M KOH in methanol,14 ml of methanol and 5 ml of toluene. Of this, 0.1 ml is used for up to2 mg of acetylated glycolipid, 0.2 ml for up to 4 mg, 0.5 ml for up to15 mg, 1 ml for up to 40 mg and so on. The sample to be deacetylated isevaporated to dryness, the appropriate amount of reagent is added andthe mixture is intermittently agitated for 2 hours after which the KOHis neutralized with acetic acid and the solvents evaporated. As anexample, deacetylation of 40 mg of acetylated glycolipid with 1 ml ofreagent produces about 1 mg of potassium acetate remaining in theglycolipid sample. If necessary, potassium ions may be removed byfiltration through e.g. chloroform/methanol-washed Amberlite® CG-50 typeI in H⁺ form (Rohm and Haas, Philadelphia, Pa., U.S.A.), and thedeacetylation mixture without added acetic acid may be filtereddirectly. However, some irreversible adsorption of glycolipid may occurand eluted resin may also contaminate. It is therefore preferred that acertain amount of potassium acetate remains in the sample.

The deacetylated plasma sample is filtered through a column of 2 g ofDEAE cellulose packed in chloroform/methanol 2:1 (by volume). The loadedsample is allowed to equilibrate for 1-2 days before elution with 50 mlof chloroform/methanol 2:1 (by volume) and 50 ml of methanol. Thepurpose of this step is to remove alkali-stable amino group-containingphospholipids which have been transferred into N-acetylated derivativesduring the acetylation procedure. This makes them acid.

A final silica gel chromatography step removes non-polar contaminantseluted in the first two fractions. The plasma sample is loaded on acolumn of 5 g of silica gel (particles smaller than 45 μm) packed inchloroform/methanol 98:2 (by volume). After elution of two fractionswith each 50 ml of chloroform/methanol 98:2 (by volume), the pureglycolipids are eluted with 50 ml of chloroform/methanol 1:3 (by volume)and 50 ml of methanol. The final yield of total non-acid glycolipids inplasma from one transfusion unit of human blood is 6-8 mg.

The preparative steps are controlled by thin-layer chromatography(silica gel 60 nanoplates; E. Merck, Darmstadt, West Germany),preferably using chloroform/methanol/water 65:25:4 (by volume) fornon-derivatized and chloroform/methanol 95:5 (by volume) for acetylatedsamples. Anisaldehyde is preferred as a detection reagent because itforms characteristic colours: glycolipids usually become green orblue-green, glycerophospholipids grey or violet, and sphingomyelin andceramide blue (cf. reference 21).

The procedure outlined above results in a total non-acid glycolipidmixture (one to about 20 sugars) completely free from non-glycolipidcontaminants. This fraction is important for testing the presence of thesecond-step receptor in various cell sources (see FIGS. 2 and 7). Toisolate the second-step receptor in pure form from such glycolipidmixtures, the final silica gel chromatographic step described above maybe slightly modified as follows. After elution with chloroform/methanol98:2 (by volume), the one-sugar second-step receptor is easily elutedwith chloroform/methanol 95:5-90:10 (by volume). For large-scalepreparation from selected sources, the procedure described above may,however, be simplified a great deal as outlined below. Accessiblesecond-step receptor sources may be mammalian intestine (see FIG. 7),yeast or fungi, and some invertebrates. For example, it has been shownthat the starfish, Asterias rubens, which is easily available from thesea, is a rich source of the second-step receptor (substance 2 in Table3, see Bjorkman et al., Biochim. Biophys. Acta 270, 1972, pp. 260-265).The mammalian brain is also a very rich source of the second-stepreceptor in the form of substance 1 in Table 3. For this purpose,lyophilized brain (e.g. bovine or porcine brain) is extracted withchloroform/methanol 2:1 (by volume) (at a volume which is 10 times theweight of the tissue) with gentle heating. The filtered extract isevaporated to dryness and reextracted with the same solvent andfiltered. The evaporated extract is subjected to alkaline methanolysisovernight (limited volume, e.g. 500 ml for one brain, of 0.2M KOH inmethanol). The mixture is then overneutralized with acetic acid to a pHof about 3.5 and solvent partitioned by adjusting the volumes tochloroform/methanol/water 8:4:3 (by volume). After standing overnight,the lower phase is evaporated to dryness and subjected to silica gelchromatography. This step is performed as a kind of filtration, using ashort, broad column packed in pure chloroform and loaded with up to 1 gof extract per g of adsorbent. The major part of the extract is elutedwith pure chloroform (cholesterol and methyl esters of fatty acids). Thesecond-step receptor, GalβCer, is eluted with chloroform/methanol95:5-90:10 (by volume). In the late elution phase, sulphated GalβCer maycontaminate. To improve the yield of GalβCer, this mixture may befiltrated through DEAE cellulose, which binds the sulphated substance.If necessary, the last traces of pigmented substances may be removed bycrystallization in ethanol. Precisely the same procedure may be appliedto lyophilized starfish. In this case, cholesterol sulphate, and notsulphated GalβCer, is removed by DEAE cellulose.

Structural analysis of isolated fractions is done on non-degradedsamples by mass spectrometry (see reference 14) and NMR spectroscopy(see reference 15) and a combined use of permethylated, permethylatedLiAlH₄ -reduced, and (in case of acid glycolipids) permethylated LiAlH₄-reduced trimethylsilylated derivatives. In this way, the sequence andanomerity of the oligosaccharide and the composition of the ceramide isobtained. Degradation is also performed to receive the information onthe type of sugars and positions of linkage, through combined gaschromatography and mass spectrometry as described in reference 16.

Several synthetic ceramides were used, including the combinationssphingosine-non-hydroxy fatty acid, sphingosine-hydroxy fatty acid,phytosphingosine-non-hydroxy fatty acid and phytosphingosine-hydroxyfatty acid (see reference 17). Similar natural combinations oflong-chain base and fatty acid were also prepared using the methodreferred to above. Sphingmyelin was obtained by the procedure describedabove. Various phosphoglycerides were prepared without alkalinedegradation and separated on DEAE cellulose into acid(phosphatidylserine and phosphatidylinositol) and non-acidphospholipids. The non-acid phospholipids were then separated on silicagel columns into phosphatidylethanolamine and phosphatidylcholine. Thesubstances were tested for purity by thin-layer chromatography andidentified by analysis of components (see reference 18).Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserinewere also purchased from Sigma Chemical Co.

Two glycoglycerolipids of plant origin, galactosyldiglyceride anddigalactosyldiglyceride, were purchased from Sigma Chemical Co.

Several synthetic monoglycosylceramides were received from Dr. IrminPascher, and their preparation has been described in Pascher, Chem.Phys. Lipids 12, 1974, pp. 303-315. These were galactopyranosylβ-andglucopyranosylβceramides with various combinations of long-chain baseand fatty acid, such as sphingosine, dihydrosphingosine,phytosphingosine and 2-hydroxy and non-hydroxy fatty acids. One of thesespecies, galactopyranosylβceramide with dihydrosphingosine and2-D-hydroxyoctadecanoic acid, was determined in its crystal conformation(Pascher et al., Chem. Phys. Lipids 20, 1977, pp. 175-191) and was usedto assign receptor binding epitope.

Chemical synthesis of the second-step receptor (GalβCer or GlcβCer) maybe performed in various ways as referred to above and reviewed in detailin reference 13. For example, dihydrosphingosine (base of formula A inFIG. 5) may be prepared according to Carter et al. or Jenny and Grob(see reference 13). Hydroxy fatty acid may be coupled to this using thep-nitrophenylester (Pascher, Chem. Phys. Lipids 12, 1974, pp. 303-315)of 2-D-acetoxyoctadecanoic acid (Karlsson et al., Chem. Phys. Lipids 12,1974, pp. 65-74). After the preparation of the 3-O-benzoyl derivative,the acetobromo derivatives of Gal or Glc may be used to prepare GalβCeror GlcβCer (substances 1 and 2, respectively, in Table 3).

Synthetic Receptor Analogues and Related Substances

Mono- or oligosaccharides coupled to a lipid part of multivalently tobovine serum albumin (substances 1-5 and 10-14 of Table 1) werepurchased from The Sugar Company, Arlov, Sweden. Novel receptoranalogues of relevance for the present invention (substances 6-9 and15-18 of Table 1) were prepared by and received from Dr. GoranMagnusson, Department of Organic Chemistry 2, University of Lund, Sweden(Dahmen et al., Carbohydr. Res. 118, 1983, pp. 292-301; Dahmen et al.,ibid. 127, 1984, pp. 27-33). The receptor analogues employed are shownin Table 1 below.

                                      TABLE 1                                     __________________________________________________________________________     Synthetic receptor analogues used for inhibition or binding                  (Sugars are of D configuration and in pyranose form)                          __________________________________________________________________________    1      Gal of GalβCETE                                                   2      Glc or GclβCETE                                                           ##STR6##                                                              4      GalβCETEBSA                                                       5      GlcβCETEBSA                                                       6                                                                                     ##STR7##                                                              7                                                                                     ##STR8##                                                              8                                                                                     ##STR9##                                                              9                                                                                     ##STR10##                                                             10                                                                                    ##STR11##                                                             11     GalβOTE                                                           12     GlcβOTE                                                           13                                                                                    ##STR12##                                                             14                                                                                    ##STR13##                                                             15                                                                                    ##STR14##                                                             16                                                                                    ##STR15##                                                             17                                                                                    ##STR16##                                                             18                                                                                    ##STR17##                                                             __________________________________________________________________________     CETE: (OCH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.2 CO.sub.2 CH.sub.3)               OTE: (OCH.sub.2 CH.sub.2 S(CH.sub.2).sub.17 CH.sub.3)                         BSA: Bovine serum albumin.                                               

Preparation of Epithelial Cells

Epithelial cells from fresh tissues were prepared in two ways: bywashing and by scraping. Washing was performed with EDTA-containingphosphate buffer in intestinal loops as described in reference 19. Aloop was filled with buffer and incubated in a water bath at 37° C. withgentle moving/stirring for a few minutes, after which the intestinalcontent was decanted off. This was repeated 5-10 times and each fractionwas centrifuged and cells examined by microscopy and marker enzyme(alkaline phosphatase, thymidine kinase) analyses. Fractions containingpure epithelial cells were pooled and used for the preparation ofglycolipids as outlined above. In this way, epithelial cells from thesmall and large intestine of several human individuals and of ratstrains were analyzed. Human small intestine was also gently scrapedwith a spoon to obtain epithelial cells somewhat contaminated withnon-epithelial residue. In a similar way, human urinary tract (urether)epithelial cells were obtained as well as epithelial cells from dog andmouse intestines. The small intestine of several other animals wasanalyzed, including cat, cod-fish, guinea-pig, hamster, hen and rabbit(see reference 20). In addition to mass spectrometry etc. forinformation on ceramide components, the samples were run on aborate-impregnated silica gel plate where monoglycosylceramides separateboth according to Gal and Glc and the major ceramide combinations ofsphingosine, phytosphingosine, non-hydroxy and 2-hydroxy fatty acid (seereference 21).

VIRUS PREPARATIONS AND ANTIBODIES

In the receptor assays used, bound virus was detected by anti-virusantibody. Polyclonal and monoclonal antibodies were prepared accordingto standard methods (Rose et al., eds., Manual of Clinical Immunology,Americ. Soc. Microbiol., Washington, D.C., 1980). The viruses testedwere known viruses which were grown and defined in professional viruslaboratories using established techniques (Lennette, ed., Manual ofClinical Microbiology, Americ. Soc. Microbiol., Washington, D.C., 1980).The specific viruses are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Viruses shown to bind to the second-step receptor                                                            Bilayer                                                                       membrane                                              Family    Member        envelope                                       ______________________________________                                        DNA viruses                                                                            Adenoviridae                                                                              Adenovirus 2 and 7                                                                          No                                                  Herpetoviridae                                                                            B95-8 EB virus                                                                              Yes                                        RNA viruses                                                                            Orthomyxo-  Influenza virus                                                                             Yes                                                 viridae                                                                       Paramyxo-   Mumps virus   Yes                                                 viridae     Sendai virus,                                                                 G variant     Yes                                                             Sendai virus,                                                                 S variant     Yes                                                 Rhabdoviridae                                                                             Rabies virus, Yes                                                             ERA strain                                                        Reoviridae  Rotavirus K8  No                                                              Reovirus 1, 2 and 3                                                                         No                                         ______________________________________                                    

Adenovirus 2 and 7 were propagated in A-549 cells and hyperimmune serawere obtained by established immunization of rabbits (Wadell, Curr. Top.Microbiol. Immunol. 110, 1984, pp. 191-220). Human rotavirus K8 waspropagated in GMK cells and a rabbit hyperimmune serum was used (Urasawaet al., Microbiol. Immunol. 25, 1981, pp. 1025-1035). Reovirus 1, 2 and3 were propagated in GMK cells and rabbit hyperimmune sera were used(Rose et al., eds., op. cit.; Lennette, ed., op. cit.). Epstein-Barrvirus was the B95-8 EB virus propagated in lymphoid monkey cells and wasprepared by Dr. L. Rymo (Rymo et al., Nucl. Acid Res. 5, 1978, pp.1387-1402), and the antibody was mouse monoclonal antibody MA providedby Dr. G. Pearson, Dep. Microbiology, The Mayo Foundation, Rochester,U.S.A. The ERA strain of rabies virus was propagated in BHK-21 cells,the antibody was mouse monoclonal antibody 101-1 (Dietzschold et al.,Proc. Nat. Acad. U.S.A. 80, 1983, pp. 70-74), and the preparations wereprovided by Dr. H. Koprowski (cf. Dietzschold et al., op. cit.). Theinfluenza virus A/PR/8/34 was propagated in embryonated hen's eggs, theantibody was mouse monoclonal antibody H2-6A5, and these preparationswere provided by Dr. W. Gerhard (Caton et al., Cell 31, 1982, pp.417-427). Influenza virus strains X-31 and X-31 HS were propagated inembryonated hen's eggs and were provided by Dr. J. C. Paulson (cf.Rogers et al., Nature 304, 1983, pp. 76-78). These strains were detectedby overlayering the thin-layer plate with a 2% suspension of humanerythrocytes (red colour for active bands) for 1 hour and washing.Influenza virus strains A/Chile/1/83, A/Philippines/2/82 andB/USSR/100/83 were obtained from SBL (Swedish State BacteriologicalLaboratory), Solna, Sweden. These viruses were also detected byoverlayering with human erythrocytes. The Z strain of Sendai virus ("S"and "G" variants; see reference 46 and Example 1) was grown inembryonated hen's eggs or in Vero cells, and the antibody was mousemonoclonal antibodies 817 and 851 (Orvell et al., J. Immunol. 129, 1982,pp. 2779-2787) and rabbit anti-Sendai antibody 121 prepared by Dr. C.Orvell. The Kilham strain of mumps virus was propagated in Vero cellsand the antibody was mouse monoclonal antibody (Orvell, J. Immunol. 132,1984, pp. 2622-2629). HTLV-I virus was prepared by Dr. J. Blomberg froman HTLV-I secreting leukemic T cell line (cf. Blomberg et al., LeukemiaRes., 1985, in press). HTLV-III was provided by Dr. R. C. Gallo,National Cancer Institute, NIH, Bethesda, U.S.A. HTLV-I and HTLV-IIIviruses were iodinated using the standard lactoperoxidase technique(Rose et al., Manual of Clinical Immunology, Americ. Soc. Microbiol.,Washington, D.C., 1980).

The antibodies were tested for the absence of unspecific binding in thebinding assay system using all ingredients except virus. In this way, itwas excluded that the antibodies bound directly to specific glycolipidbands, which phenomenon otherwise may produce falsely positive results.

Assays for Virus Binding Specificity and Estimation of Relative BindingStrength

The assay for the detection of virus binding to glycolipids and fortesting of detailed specificity of binding has been developed by thepresent inventors and is of decisive importance for the invention (cf.Hansson et al., FEBS Lett. 170, 1984, pp. 15-18). In principle, thevirus to be assayed is layered on a chromatogram with separatedglycolipids from target cells or other sources and allowed to interactwith potential receptor substances. After careful washings, bound virusis detected by anti-virus antibody and radiolabelled anti-anti-bodyfollowed by autoradiography. In some cases, the virus particle wasdirectly labelled before binding. The detailed procedure is as follows:

Mixtures of total lipids (up to 100 μg in each lane) or totalglycolipids (20-40 μg in each lane) or pure glycolipids (0.01-1 μg) wereseparated on aluminum sheets, about 5×5 cm, coated with silica gel 60(Merck), usually with chloroform/methanol/water (60:35:8, by volume) asthe solvent for non-acid glycolipids, and with chloroform/methanol/2.5Mammonia (60:40:9, by volume) as the solvent for acid glycolipids. Forpurposes of comparison, a parallel plate is detected chemically byspraying and heating with anisaldehyde solution. For virus binding, thedried chromatogram with separated substances is dipped for 1 minute in200 ml of diethylether containing 0.5% (w/v) of polyisobutylmethacrylate(Plexigum P28, Rohm GmbH, Darmstadt) and dried for 2 minutes. The plateis then sprayed with phosphate-buffered saline (PBS) of pH 7.3containing 2% bovine serum albumin (BSA) and 0.1% NaN₃ (solution A) andthen immersed in solution A and placed in a Petri dish for 2 hours.After tipping off solution A, the virus suspension is added (about 25 μgper ml with about 2 ml for a plate of the dimensions given above) to thechromatogram placed horizontally in the humidified atmosphere of a Petridish. After incubation for 2 hours, the virus suspension is tipped offand the plate is washed six times with PBS, 1 minute each time. In atypical case of antibody, monoclonal antibody 817 directed againstSendai virus produced in ascitic fluid is diluted 1:100 with solution A,using about 2 ml per plate, with incubation for 2 hours. After washingfive times with PBS, about 2 ml of rabbit anti-mouse Fab is incubatedfor 2 hours (4×10⁵ cpm/ml of ¹²⁵ l-labelled F(ab')₂, the RadiochemicalCentre, Amersham). After six washings in PBS, the plate is dried andexposed to XAR-5 X-ray film (Eastman), usually for 2-3 days, using anintensifying screen.

The treatment with plastic produces a hydrophobic surface. Separatedglycolipid or other bands are thus induced to be exposed on thehydrophobic solid surface similar to the way lipids are exposed in thebiological membrane. This means that the test substance is denselyanchored with its paraffin chains in the plastic surface with the polarhead groups exposed and accessible to the environment. This mimics thesurface monolayer of the living cell. This plastic treatment is highlycritical for specificity and reproducibility and explains the advantageof this solid-phase method over traditional inhibition based on"solubilized" aggregates or micelles (see reference 22).

The detection limit varies with the avidity of the ligand but is in therange of 5-50 ng of receptor, or in about the same picomole range. For areceptor candidate to be considered negative in the tabulated results,there should be no darkening at a one or more microgram level. Goodbinders give saturating black bands at 100 ng. An obvious advantage ofthis assay is that mixtures of substances are first separated intosubstance species, avoiding the risk of shielding of minor components,or false negative binding due to contaminating substances. Also, thecoating with albumin blocks unspecific hydrophobic sites, whichotherwise may cause false positive results. Finally, the extensivewashings remove more loose unspecific associations. By comparison,traditional inhibition assays usually incubate virus with target cellsin suspension in the absence or presence of sonicated micelles. In caseof the hemolysis assay, simple photometry is done on the mixture aftercentrifugation (cf. Huang. Lipids 18, 1983, pp. 489-492). Thus, noalbumin is present, and there are no washing steps analogous to thepresent assay.

For quantification of virus binding, a technique was adopted from theanalogous solid-phase binding or antibodies to microtiter wells(Brockhaus et al., J. Biol. Chem. 256, 1981, pp. 13223-13225). Adilution series of glycolipid or other substances in 50 μl of methanolis allowed to evaporate in the microtiter well overnight at roomtemperature. 100 μl of 2% BSA in PBS are then incubated for 2 hoursafter which the well is rinsed once with this volume and solution. 50 μlof a suspension of 1.5 μg of virus in BSA-PBS is incubated for 4 hours,followed by four washings with 100 μl each of BSA-PBS. In case of Sendaivirus, 50 μl of ascitic fluid-produced antibody 817 diluted 1:100 insolution A is incubated for 4 hours followed by four washings. Finally,50 μl of rabbit anti-mouse Fab (2.5×10⁴ cpm ¹²⁵ l-labelled F(ab')₂, theRadiochemical Centre, Amersham) is incubated overnight at 4° C. followedby five 100 μl washings with BSA-PBS. The wells are cut from the plateand assayed individually for ¹²⁵ l in a spectrometer.

Inhibition Studies

Attempts to inhibit the binding of virus by means of the two assaysdescribed above were done by a preincubation of the virus for one or twohours with up to 2 mg per ml of free saccharide or BSA-conjugatedsaccharide in PBS or BSA-PBS. This mixture is then added in toto to theplate or well.

Inhibition studies were also performed by means of an ELISA technique(enzyme-linked immunosorbent assay) as follows (see reference 1 and Roseet al., eds., Manual of Clinical Immunology, Americ. Soc. Microbiol.,Washington D.C., 1980). In this case, microtiter wells (type Cooks M 29)were coated with glycolipid in methanol as described above, or withrabbit anti-Sendai antiserum as a standardized control (see reference1). The glycolipids used were GalβCer, which is a virus binder, seeglycolipid 1 to Table 3 (about half of the fraction containednon-hydroxy fatty acid and this species is inactive), and globoside,which is a non-binder, see glycolipid 16 of Table 3, both at 500 ng perwell. 50 μg/ml of antiserum in sodium bicarbonate buffer, pH 9.6 (100μl/well) were added. The wells were then saturated for 2 hours at 37° C.with 2% BSA in 50 mM Tris-HCl and 0.15 M NaCl buffer, pH 8.5, and thenwashed twice with this buffer containing 1% BSA. 1.5 μg per well ofSendai virus in 100 μl of the 1% BSA buffer were incubated for 2 hours,followed by four washings with 100 μl each of the 1% BSA buffer.Detection of bound virus was carried out by incubation for 1.5 hours at37° C. with 100 μl of antibody 851 diluted 1:100 with buffer, followedby four washings with 100 μl each of the buffer. 100 μl of horseradishperoxidase-conjugated rabbit anti-mouse antibody (Dakopatts, Glostrup,Denmark; P 161, 10 mg/ml diluted 1:500) were incubated for 1.5 hours at37° C. followed by four washings with buffer. 100 μl per well of OPDsolution (4 mg of orthophenylenediamine, Sigma, dissolved in 10 ml of0.1M sodium citratephosphate buffer, pH 5.0 to which had been added 4 μlof 30% H₂ O₂) were incubated for 15 minutes, and then the reaction wasstopped by adding 50 μl of 0.5M H₂ SO₄ per well. Optical reading wasperformed at 492 nm. Typical values were as follows: all steps exceptvirus=0.150; binding to globoside=0.200; binding to GalβCer=0.650;binding to antiserum=1.7.

Inhibition of virus binding by substance 9 of Table 1 was performed asfollows. About 2 μg of virus were preincubated in a siliconized testtube with about 200 μg of substance 9 for 2 hours at 37° C. and thentransferred in 50 μl portions to the wells and incubated for 1 hour at37° C., followed by four washings with the buffer containing 1% BSA. Thesubsequent steps were carried out as described above. Substance 9 wasdiluted 1:4 in 7 separate preincubation steps.

Preliminary experiments were also performed on the inhibition of virusbinding in the two assays described by preincubating the virus as abovewith sonicated micelles of amphipathic glycolipids and other substances.This was done for Sendai virus. Plaque inhibition test (Lycke et al.,eds., Textbook of Medical Virology, Butterworths, London, 1983) was donewith the ERA-similar virus strain CVS-3337 as follows:

Phosphatidylcholine (200 μg), cholesterol (100 μg) and Galβ1→4GlcβCerwith 2-hydroxy fatty acid (230 μg) or the first two lipids andphosphatidylserine (200 μg) were dissolved in chloroform/methanol andthe solvent evaporated in a tube. 4 ml of PBS were added and the mixturesonicated in a Vetrasonics model W-370 for 1/2 hour at room temperature.The virus was diluted with PBS to give approximately 4×10³plaque-forming units per ml. 0.5 ml virus and 0.5 ml sonicated micelles(or PBS as control) were incubated for 3 hours at room temperature.Dilutions and titration of remaining infectious virus on CER cellmonolayers are carried out under standard conditions (Lennette, ed.,Manual of Clinical Microbiology, Americ. Soc. Microbiol., WashingtonD.C., 1980).

EXAMPLE 1 Discovery of the Second-Step Receptor

Sendai virus was used as the model virus for analysis (cf. the overlayassay method described in Materials and Methods) because it isrelatively well studied with respect to its binding to neuraminicacid-containing receptors (the first-step receptor) (see reference 26)(FIGS. 1 and 2 illustrate the binding of the virus to the conceptualfirst-step and second-step receptors, respectively). Preparations of thevirus prepared as described in Materials and Methods were analyzed, andtwo previously unknown receptor binding variants of the Z strain wereshown to carry separate specificities in the case of binding toneuraminic acid-containing glycolipids (gangliosides). The S variantbound to gangliosides of human erythrocytes only but not to gangliosidesof the human brain (FIG. 1). The G variant, on the other hand, bound togangliosides of both sources (not shown). It is known that thegangliosides of the two sources are structurally related but differ intheir core sequences. Thus, cleavage of neuraminic acid by the enzymeneuraminidase from the gangliosides completely abolishes binding of thetwo virus preparations. This shows that the binding to gangliosides iscompletely dependent on the presence of neuraminic acid but that thebinding epitope of the glycolipids also includes the non-neuraminic acidparts, which differ between brain and erythrocyte. Therefore, thefirst-step receptor specificity in these two cases differ and shouldcorrespond to separate, chemically different sites on the two virusesrecognizing the receptors. That several bands in the assay may bind isexplained by the presence of the receptor epitope on core substances ofvarious sizes which differ in chromatographic mobility. The fact thatthe G variant of the virus binds to gangliosides of both origins may beexplained by a less strict specificity in the binding compared to the Svariant.

In contrast to the different binding to gangliosides (first-stepreceptors), the two variants bind the second-step receptors in anidentical way. This is shown for the S variant in FIG. 2. As will beexplained in further detail in Example 2, this binding is to glycolipidslacking neuraminic acid and is thus chemically distinct from thefirst-step binding.

This shows that a virus may have two chemically distinct and separatereceptors. Furthermore, the results obtained from the assay illustratethe specificity of the method as explained in the specification and inMaterials and Methods. The selective binding is evident when comparingthe patterns obtained by autoradiography (virus binding) with chemicalspraying (all substances visualized). Some major bands appearing fromspraying and being structurally closely related to receptor substancesare completely negative for virus binding (cf. Example 2).

EXAMPLE 2 Analysis of Natural Receptor Candidates for BindingSpecificity with Respect to Several Viruses

A large number of receptor candidates (Table 3) was analyzed for bindingactivity using the overlay assay described in Materials and Methods.

                                      TABLE 3                                     __________________________________________________________________________    No.                                                                              Glycolipid structure          Virus binding                                __________________________________________________________________________     1 GalβCer                  +                                             2 GlcβCer                  +                                             3 Galβ1→4GlcβCer                                                                             +                                             4 Galα1→4GalβCer                                                                            +                                             5 Galα1→3Galβ1→4GlcβCer                                                         +                                             6 Galα1→4Galβ1→4GlcβCer                                                         +                                             7 Fucα1→2Galβ1→4GlcβCer                                                         +                                             8 GlcNAcβ1→3Galβ1→4GlcβCer                                                       +                                             9 GalNAcβ1→4Galβ1→4GlcβCer                                                       +                                            10 NeuAcα2→3Galβ1→4GlcβCer                                                       -                                            11 Galα1→3(Fucα1→2)Galβ1→4GlcβC       er                            +                                            12 GalNAcα1→3(fucα1→2)Galβ1→4Glc.bet       a.Cer                         -                                            13 Galα1→3Galα1→4Galβ1→4GlcβCer       .                             -                                            14 Galβ1→3GlcNAcβ1→3Galβ1→4GlcβCe       r                             +                                            15 Galβ1→4GlcNAcβ1→3Galβ1→4GlcβCe       r                             +                                            16 GalNAcβ1→3Galα1→4Galβ1→4GlcβC       er                            -                                            17 Galβ1→3GalNAcβ1→4Galβ1→4GlcβCe       r                             -                                            18 Fucα1→2Galα1→3Galα1→4Galβ1.       fwdarw.4GlcβCer          -                                            19 Fucα1→2Galβ1→3GlcNAcβ1→3Galβ        1→4GlcβCer        -                                            20 Fucα1→2Galβ1→4GlcNAcβ1→3Galβ1       →4GlcβCer         -                                            21 Galβ1→3(Fucα1→4)GlcNAcβ1→3Gal.beta       .1→4GlcβCer       +                                            22 Galβ1→4(Fucα1→3)GlcNAcβ1→3Gal.beta       .1→4GlcβCer       +                                            23 NeuAcα2→3Galβ1→4GlcNAcβ1→3Gal.beta       .1→4GlcβCer       -                                            24 Fucα1→2Galβ1→3(Fucα1→4)GlcNAc.bet       a.1→3Galβ1→4GlcβCer                                                                 -                                            25 Fucα1→2Galβ1→4(Fucα1→3)GlcNAc.bet       a.1→3Galβ1→4GlcβCer                                                                 -                                            26 Galβdiglyceride          +                                            27 Galα1→6Galβdiglyceride                                                                    +                                            __________________________________________________________________________     1-25: Animal origin, 26-27 plant origin. The monosaccharides are of D         configuration except Fuc which is L, and are all in pyranose form.            + indicates binding and - indicates lack of binding.                     

Mixtures of extracts from target cells or tissues for viral infectionsextracted by means of organic solvents as described in Materials andMethods were pre-separated on the chromatogram surface allowing thedetection of active substances after overlaying with the viruspreparation in question. A binding substance was isolated andstructurally characterized and used in pure form for more detailedbinding studies. Sendai virus was first used in these analyses. As onesingle overlay assay may contain more than 100 substances of a diversestructure, a very efficient selection of actual receptors was obtained.Using human and animal tissues of various kinds like nerve tissue,blood, gastrointestinal tract, urinary tract, and including both adultand fetal tissues, only the glycolipids marked with a + in Table 3 andfurther defined in Table 4 were shown to be receptors. There was nobinding at relevant levels of other surface membrane or other substanceslike cholesterol or various glycerophospholipids that were coextractedin the procedures employed. On the other hand, several of the virusesanalysed (cf. Table 2), including Sendai virus (cf. Example 1), showed aglycolipid binding different from that of Tables 3 and 4 which waschemically distinct and corresponded to the first-step receptor.

An important finding was that the binding to the natural receptorsdepends on the composition of the lipophilic part of the glycolipid, theceramide, as defined in Table 4 and illustrated for some species in FIG.3. Only species with 2-D-hydroxy fatty acid in the ceramide are active.Therefore, all natural glycolipids used for detailed testing concerningthe importance of variation in the carbohydrate part (Table 3) werechosen for the presence of 2-hydroxy fatty acid. Species referred to inTable 3 as being positive are active when containing 2-hydroxy fattyacid but inactive when containing non-hydroxy fatty acid.

The virus is able to bind glycolipids with up to five sugars (Table 3).However, quantitative binding studies (cf. Materials and Methods) revealthat the one-sugar substances GalβCer and GlcβCer are the best binders.In FIG. 4, curve A represents these two glycolipids. Curve C representsthe two-sugar glycolipid 3 of Table 3 and curve D represent thethree-sugar glycolipid 5. It is important to note that the avidity(strength) of binding for this second-step receptor is on the same orderof magnitude as that of the established first-step receptor (curve B).The binding to larger glycolipids is therefore explained by arecognition of an internal part of the molecules. Such an unconventionalbinding is not unexpected in view of the inventors' results fromcarbohydrate receptor binding for several bacteria and bacterial toxins(see reference 23, 25 and 46). The fact that some (the major part) ofthe glycolipids tested do not bind although they contain the bindingepitope, e.g. →4GlcβCer, is rationalized by a steric hindrance in theaccess of the large virus particle to this one-sugar epitope. It is alsofurther evidence for a stereospecificity at the molecular level,allowing the use of the concept of receptor specificity, and stronglyarguing against an unspecific interaction.

Of interest to wider applications, plants are known to contain receptorglycosphingolipids (see reference 41). Substance 2 of Table 3 with2-hydroxy fatty acid is a dominating glycolipid in plant leaves. Also,substances 26 and 27 shown to bind Sendai virus are relatively abundantin plants and may serve as receptors for plant viruses.

The finding that there is no binding to free ceramide (lackingcarbohydrate) is important for the interpretation of the binding epitopeof the receptor substances. Furthermore, in the assay, there is noinhibition of binding of virus to receptors when adding various freeoligosaccharides or oligosaccharides, e.g. Galβ, Glcβ or Galβ1→4Glcβ,coupled multivalently to albumin (cf. Example 6). From this it may beconcluded that there is binding to the glycolipid only; neither ceramidealone nor saccharide alone (or coupled to a protein) is able to interactwith the virus.

The curves obtained from the quantitative binding studies (FIG. 4)indicate that the avidity of the binding is very similar for thefirst-step and second-step receptors (curve B and A, respectively). Asthe first-step receptor has already been studied for biologicalrelevance (see reference 3), this means that the binding avidity for thesecond-step receptor is at a biologically adequate level. Also, the bestsecond-step binder is that with one sugar (compare curves A, C and D),and some binders with several sugars do not raise up (curve E) althoughthe binding is clearly detectable in the overlay assay (Table 3).

A second conclusion from quantitative analyses by means of this assay isthat the binding is of the low-affinity type needing multivalency to beefficient. This is based on a comparison, in the same assay, of virusbinding with the binding of several bacteria and bacterial toxins totheir respective receptor glycolipids. Several bacteria (see reference23 and 46) and the Shiga toxin (see reference 25 and 46) bind similarlyto the virus (the curves raise at similar levels of receptor dilution).On the other hand, in case of cholera toxin (similar size as Shigatoxin), the raise of the curve is shifted to the left at a 10² -10³greater receptor dilution. These different locations of the curvescoincide with the possibility to inhibit ligand binding to the receptorglycolipids using soluble, univalent oligosaccharide receptor analogues.Cholera toxin is easily inhibited but for Shiga toxin, there is nodetectable inhibition at the level of 5 mg/ml of the receptordisaccharide. However, the Shiga toxin is easily inhibited using thedisaccharide coupled multivalently to albumin. The conclusion is ahigh-affinity binding in case of cholera toxin and a low-affinitybinding in case of the Shiga toxin, the latter needing multivalency tobe efficient. A similar situation as for Shiga toxi exists for severalbacteria. As the curves for virus binding coincide with those of Shigatoxin and several bacteria but not with that of cholera toxin, the virusbinding is of the low-affinity type. Therefore, application of thenatural binding epitope for virus binding would seem to need amultivalent presentation. This is present in the binding assays and inthe biological membrane (Example 4) and corresponds to a multiplicity ofbinding proteins on the virus surface.

The approach described above for Sendai virus was extended to a seriesof other viruses, representing most of the known virus families (Table2). All viruses shown in Table 2 were shown to bind in a way which wasvery similar to Sendai virus in the case of the second-step receptor,although several of them also showed a separate first-step receptoranalogous to that of Sendai virus (Example 1). The analyses of theseviruses was performed using the virus preparations and antibodies fordetection as described in Materials and Methods. To cover as manyreceptor candidates as possible, the glycolipid mixtures employed forthe assay shown in FIG. 2 were used. Additionally, several isolatedsubstances of Table 3 were used in dilutions on the thin-layer plate totest the avidity of binding compared to Sendai virus. The results showedthat all viruses tested had a binding pattern identical with or verysimilar to that of FIG. 2, with similar binding avidity. This means thatthe viruses tested have a specific binding property in common whichshould correspond to a common binding protein on the virus surface. Thisproperty does not depend on the different envelopes of the viruses orthe character of the genome (Table 2). Therefore, morphologically andgenetically different viruses causing a wide variety of human and animaldiseases affecting separate organ systems possess a common bindingproperty, namely a second-step receptor which is probably used forpenetration.

EXAMPLE 3 Chemical Characterization of Receptors and Analysis of theIdeal Conformation of the Binding Epitope

As described in Materials and Methods, individual receptor-activesubstances were isolated in pure form and chemically characterized. Thisgave information both on the structure of the polar/hydrophobic part(i.e. the saccharide structure including type of sugars, sequence,position of binding, anomerity and ring size) and the non-polar part(i.e. the ceramide, including fatty acid chain structure as regardstheir length, branching and degree of unsaturation and the position andconfiguration of the hydroxyl groups, and the long-chain base structureas regards the length of the paraffin chain, unsaturation, and theposition and configuration of functional groups). The methods used werehigh technology mass spectrometry (see reference 14) and NMRspectroscopy (see reference 15) in addition to conventional degradationmethods. To get a wide range of structures for testing, tissues fromdifferent animals were used as preparative sources (see reference 13aand 33). Some of the epithelial tissues used have been describedconcerning glycolipid structures such as human intestine (see reference42 and 43), rat intestine (see reference 13a), mouse intestine (seereference 20 and 44), dog intestine (see reference 45), and intestinesfrom cat, cod-fish, guinea-pig, hamster and rabbit (see reference 20).Several other tissues were alos analyzed as described in Example 2. Manyof these structures have recently been reviewed (see reference 13). Thismeans that a very large number of glycolipids and other substances notincluded in Table 3 were analyzed but found negative for binding toSendai virus.

A very important feature is the dependence of binding on the ceramidestructure, as summarized in Table 4 for one particular glycolipid, No. 3of Table 3, isolated from the indicated natural sources and carefullyanalyzed for its detailed structure. The experiments were performed asdescribed in Materials and Methods using the overlay binding assay.Criteria for positive binding were also defined. The glycolipids testedwere defined by the structural methods described in Materials andMethods. The detailed structures of various molecular species ofceramides are shown in FIG. 5. The letters in Table 4 also refer to thisFigure.

                  TABLE 4                                                         ______________________________________                                        Effect on virus binding of the ceramide structure of the glycolipid           Combination of fatty acid                                                                        Source of     Virus                                        and long-chain base                                                                              preparation   binding                                      ______________________________________                                        B: Non-hydroxy fatty acid-                                                                       Human erythrocytes                                                                          -                                            sphingosine                                                                   C: Non-hydroxy fatty acid-phyto-                                                                 Dog small intestine                                                                         (+)                                          sphingosine                                                                   D: 2-Hydroxy fatty acid-sphingosine                                                              Dog small intestine                                                                         ++                                           E: 2-Hydroxy fatty acid-phyto-                                                                   Rat small intestine                                                                         ++                                           sphingosine                                                                   ______________________________________                                         -: no binding                                                                 (+): weak binding                                                             ++: good binding                                                         

Thus, although the oligosaccharide structure is identical for the fourvariants, only those having a 2-D-hydroxy fatty acid in the ceramidebind the virus. This was repeatedly reproduced for other glycolipids ofTable 3 as well. This, at first sight, appears to mean that the2-hydroxy group is specifically involved in the binding. However,binding studies of various synthetic glycolipids rule this out (Example5). Instead, the interpretation is that glycosphingolipids with a2-hydroxy fatty acid differ in conformation from glycosphingolipidshaving a non-hydroxy fatty acid. This has been documented by X-raycrystallographic analysis of the former (see reference 31) and NMRanalysis of the latter (see reference 32). In these cases, syntheticone-sugar glycosphingolipids were used. The 2-hydroxy glycosphingolipidhas a spoon-like or shovel-like conformation (cf. FIG. 6) with thesaccharide ring positioned at an angle of about 110° in relation to theceramide. In the non-hydroxy isomer, this angle is about 180°. Goingthrough all other structural parameters of the substances analyzed, thisis the only common denominator of interpretation. Therefore, asdiscussed above, the importance of the 2-hydroxy group of theglycosphingolipid is to present the first saccharide in the proper bentpreferred conformation. This makes the α side of this sugar accessiblefor binding, which has therefore been interpreted as part of the bindingepitope. In the presentation of the sugar (more or less at right anglesto the membrane-like surface of the assay) in the case of thenon-hydroxy glycosphingolipids, this binding epitope is not likely to beaccessible for virus binding.

EXAMPLE 4 Receptors on Target Cells at the Port-of-Entry of ViralInfections, and on Certain Other Cells

As described in Examples 2 and 3, a large number of epithelial tissuesof different animals have been used by the present inventors as sourcesfor the preparation of glycolipids. To test more precisely the locationof receptor glycolipids to the epithelial cells which are the directtargets of viral infections, such cells were isolated as described inMaterials and Methods. The technique was worked out for rat intestinalcells (see reference 19) including both small and large intestine, andwas applied also on human small and large intestine. Also, a mucosascraping was performed on human intestine (see reference 42) and humanurether. A common denominator of all epithelial samples analyzed (cf.Materials and Methods), regardless of tissue or animal source, is theexistence of GlcβCer (substance 2 of Table 3 with 2-hydroxy fatty acid)and in many cases also GalβCer with 2-hydroxy fatty acid. Therefore, themost optimal natural Sendai virus binders (curve A of FIG. 4) existgenerally in epithelial cells. As an illustration, FIG. 7 shows theresult from an analysis of epithelial cells of small intestine of fourhuman individuals of different blood group phenotypes. While, asexpected, the slow-moving blood group fucolipids vary between thesamples, they have the receptor-type glycolipids in common. Therefore,all cells which the virus invades when infecting an organism contain thesecond-step receptor.

Non-epithelial cells have much lower amounts or may practically lackthese receptor glycolipid species. Contrary to what has previously beenheld to be the case, erythrocytes used for hemolysis (penetration assay)contain the receptors in that small but definite amounts of GalβCer andGlcβCer with 2-hydroxy acid have been found on human, bovine and porcineerythrocytes.

The general structural characteristics of the epithelial surfacemembrane of which the receptor glycolipids are an important part arerelevant in the present context. A specific feature of the sphingolipidwith its varying number of hydroxyl groups of the ceramide is thecombined hydrogen bond donor and acceptor capacity (FIG. 8) making upone of three defined zones of the membrane (see reference 33). This isprobably of decisive importance to membrane stability which depends on alaterally oriented network of hydrogen bonds. The data on the cellsurface membrane available at present make it seem likely that the virushas developed a binding property which interferes with this stabilitythrough specific interaction with all three parts of an essentialcomponent of this network.

EXAMPLE 5 Study of the Binding of Sendai Virus to Non-BiologicalSynthetic Substances which are Analogues of the Natural Receptor

A number of synthetic substances shown in Table 1 were analyzed forvirus binding in the two assays described in Matarials and Methods.Table 5 summarizes the semiquantitative binding results fromoverlayering of Sendai virus to dilutions of the substance in question.The + signs do not correspond to those of Table 3 or 4 but are only forrelative comparison within Table 5.

As shown, there is weak binding to No. 12, but not to Nos. 11, 13 or 14,indicating some preference for Glcβ. The two-chain compounds are betterbinders (compare 15 with 13). However, most importantly, the sulphonoanalogues, Nos. 16-18, bind most strongly, the two-chain compounds beingpreferred. A more precise quantification by means of the quantitativebinding assay described above shows that No. 18 has the same avidity asthe corresponding natural receptor, No. 3 to Table 3, both representedby curve C in FIG. 4. These substances were designed on the basis of theresearch done on the natural receptors, and it is interesting to notethat the properties and dimensions of the natural epitope (Example 3)and the specifications of receptor analogues are completely satisfiedwith these substances. Thus, the requirements to parts A, B and C (asexplained above) and their relative dimensions are met although part Bis non-chiral and carries one or two sulphono groups instead of a chiralpart with an amide linkage and two hydroxyls. Evidently, the α side ofthe sugar is available for binding, which is the requirement outlined inExample 3. For these analogues, this is explained by the sulphono groupsgiving four only sites which by repulsion and possibly hydrogen bondingto water produce a much less dense packing of the molecules in themonolayer of the assays.

It further appears from Table 5 that several other synthetic substancesdo not bind the virus. This is due either to incomplete fulfilment ofthe requirements for B (substances Nos. 11, 13 and 14 which may becompared with substances Nos. 1, 3 and 4, respectively, of Table 3,which are good binders), or to close chain packing making thepresentation of the αside of A impossible in the absence of aconformation-determining factor like the 2-hydroxy fatty acid of thenatural receptor.

                                      TABLE 5                                     __________________________________________________________________________     Synthetic receptor analogues used for inhibition or binding                  (Sugars are of D configuration and in pyranose form)                          __________________________________________________________________________                                        Inhibition                                1  Gal or GalβCETE             -                                         2  Glc or GclβCETE             -                                             ##STR18##                       -                                         4  GalβCETEBSA                 -                                         5  GlcβCETEBSA                 -                                         6                                                                                 ##STR19##                       -                                         7                                                                                 ##STR20##                       -                                         8                                                                                 ##STR21##                       -                                         9                                                                                 ##STR22##                       +                                         10                                                                                ##STR23##                       -                                                                             Binding                                   11 GalβOTE                     -                                         12 GlcβOTE                     (+)                                       13                                                                                ##STR24##                       -                                         14                                                                                ##STR25##                       -                                         15                                                                                ##STR26##                       +                                         16                                                                                ##STR27##                       ++                                        17                                                                                ##STR28##                       +++                                       18                                                                                ##STR29##                       +++                                       __________________________________________________________________________     CETE: (OCH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.2 CO.sub.2 CH.sub.3)               OTE: (OCH.sub.2 CH.sub.2 S(CH.sub.2).sub.17 CH.sub.3)                         BSA: Bovine serum albumin.                                                    -: no binding/inhibition; (+): weak binding; +: binding; ++: good binding     +++: excellent binding.                                                  

EXAMPLE 6 Inhibition Experiments

Gal, Glc and Galβ1→4Glc in free form or derivatized or coupled to BSA(substances 1-6 and 10 to Table 5) were used in attempts to inhibitSendai virus binding to receptors using mixtures of glycolipids as shownin FIG. 2, but also pure substances Nos. 1, 2 and 3 of Table 3 onthin-layer plates by preincubating the virus with the sugars beforelayering on the plate. There was no tendency of weakening of theautoradiographic spots, indicating that these substances were incapableof competing with the receptors for binding. The conclusion is that thecarbohydrate part of the natural receptor substances in univalent ormultivalent form is not in itself able to bind to the virus. Similarly,there was no inhibition in the microtiter assay using substance 3 ofTable 3 as the receptor.

Preincubating Sendai virus wiht sonicated micelles of various membranelipids gave a clear inhibition of virus binding on thin-layer plates forseveral glycolipids of Table 3. Also, in the plaque inhibition test, arabies virus strain was shown to be completely inhibited by carefullysonicated liposomes containing substance 3 of Table 3. At similardilution in the titration, liposomes containing phosphatidylserine gaveno reduction of plaques.

Preincubation of Sendai virus with soluble receptor analogues(substances 7-9 of Table 5) before overlaying of virus on a thin-layerplate, or incubation in wells, gave the following results.

Sendai virus (60 μg) and substance 9 (1 mg) in 2 ml of PBS werepreincubated for 1 hour at room temperature before overlayering on athin-layer plate and incubation and working up as described in Materialsand Methods, followed by autoradiography to detect bound virus. Theglycolipid samples applied to the plate were total gangliosides of humanerythrocytes (first-step receptor, see Example 1), and glycolipids 1, 2,3 and 5 of Table 3, in addition to several suitable negative controls.Compared to the control plate (binding of virus without preincubationwith substance 9 of Table 5), there was a significant reduction of virusbinding after preincubation with substance 9, evaluated on the basis ofthe dark areas on the autoradiogram becoming lighter. Thus, binding tothe glycolipids 1, 2 and 3 was reduced several times and the band forglycolipid 5 disappeared altogether. On the contrary, there was noeffect on the binding to the first-step receptor (erythrocytegangliosides, compare FIG. 1). Similar preincubations with 1 mg/ml ofsubstances 7 and 8 gave no visible inhibition of virus binding. Theconclusion from these experiments with the overlay assay is that thesynthetic receptor analogue in multivalent form (substance 9 of Table5), but not in univalent form (substances 7 and 8 of Table 5), is ableto produce a selective inhibition of binding to the second-step receptorwithout having any effect on binding to the first-step receptor.

Preincubations of Sendai virus with these soluble receptor analoguesusing microtiter wells and an ELISA assay (see Materials and Methods)gave the following results. Preincubation with substance 9 of Table 5gave a reduction of virus binding to the second-step receptor GalβCer(A₄₉₂ =0.350 as a typical value at the highest concentration with areturn to the maximum level after three 1:4 dilutions, see Materials andMethods). There was no inhibitory effect on the binding of virus tocoated antiserum and there was no inhibition by preincubation withunivalent analogue (substances 7 and 8). The results therefore coincidewith those from the overlay assay above with a selective inhibition ofbinding to the second-step receptor, but not to the antibody, and a needfor multivalency in order for the analogue to be effective.

BIBLIOGRAPHY

1. Lycke et al., eds., Textbook of Medical Virology, Butterworths,London, 1983.

2. Lycke et al., see 1. White et al., Quart. Rev. Biophys. 16, 1983, pp.151-195.

3. Lonberg-Holm et al., eds., Virus Receptors, part 2, "Animal Viruses,Receptors and Recognition", ser. B, vol. 8, Chapman and Hall, London,1981. Dimmock, J. Gen. Virol. 59, 1982, pp. 1-22.

4. Bell, Biophys. J. 45, 1984, pp. 1051-1064.

5. Lycke et al., see 1.

6. MacDonald et al., Virology 134, 1984, pp. 103-117.

7. Schlegel et al., Cell 32, 1983, pp. 639-646. Superti et al., Arch.Virol. 81, 1984, pp. 321-328.

8. Huang, J. Gen. Virol. 64, 1983, pp. 221-224. Huang, Lipids 18, 1983,pp. 489-492.

9. Paulsen, Chem. Soc. Rev. 13, 1984, pp. 15-45.

10. Lee et al., in Horowitz, ed., The Glycoconjugates, Vol. 4, AcademicPress, New York, 1982, pp. 57-87. Dahmen et al., Carbohydr. Res. 127,1984, pp. 27-33.

11. Lycke et al., see 1.

12. Middlebrook et al., Microbiol. Rev. 48, 1984, pp. 199-221.

13. Kanfer et al., eds., Sphingolipid Biochemistry, Handbook of LipidResearch, Vol. 3, Plenum Press, New York, 1983.

13a. Breimer et al., J. Biol. Chem. 257, 1982, pp. 557-568.

14. Breimer et al., in Quayle, ed., Adv. Mass Spectrom., Heyden and SonsLtd., London, Vol. 8B, 1980, pp. 1097-1108.

15. Falk et al., Arch. Biochem. Biophys. 192, 1979, pp. 164-202.

16. See Kanfer et al., 13.

17. Karlsson et al., J. Lipid Res. 12, 1971, pp. 466-472.

18. Karlsson et al., Eur. J. Biochem. 46, 1974, pp. 243-258.

19. Breimer et al., Exp. Cell Res. 135, 1981, pp. 1-13.

20. Breimer et al., J. Biochem. 90, 1981, pp. 589-609.

21. Karlsson et al., Biochim. Biophys. Acta 306, 1973, pp. 317-328.

22. see Huang, Lipids, ref. 8.

23. Hansson et al., in Chester et al., eds., Glycoconjugates, Proc. 7thInt. Sympos., Rahms, Lund, Sweden, 1983, pp. 631-632.

24. Magnusson et al., in Chester et al., eds., Glycoconjugates, Proc.7th Int. Sympos., Rahms, Lund, Sweden, 1983, pp. 643-644.

25. Brown et al., in Chester et al., eds., Glycoconjugates, Proc. 7thInt. Sympos., Rahms, Lund, Sweden, 1983, pp. 678-679.

26. see ref. 3.

27. see 23 and 25.

28. see Dimmock, ref. 3.

29. see ref. 23.

30. see ref. 25.

31. Pascher et al., Chem. Phys. Lipids 20, 1977, pp. 175-191.

32. Skarjune et al., Biochemistry 21, 1982, pp. 3154-3160.

33. Karlsson, in Chapman, ed., Biological Membranes, Academic Press,London, Vol. 4, 1982, pp. 1-74.

34. see White et al. in ref. 2. see Dimmock in ref. 3.

35. Richardson et al., Virology 131, 1983, pp. 518-532.

36. see White et al., ref. 2.

37. see ref. 35.

38. Sebesan et al., Can. J. Chem. 62, 1984, pp. 1034-1045.

39. see ref. 31.

40. see ref. 33.

41. Hitchcock et al., eds., Plant Lipid Biochemistry, Academic Press,London, 1971.

42. Falk et al, FEBS Lett. 101, 1979, pp. 273-276.

43. Bjork et al., in Cartron et al., eds., Red Cell MembraneGlycoconjugates and Related Genetic Markers, Libraire Arnette, Paris,1983, pp. 125-137.

44. Hansson et al., FEBS Lett. 139, 1982, pp. 291-294.

45. Hansson et al., Biochim. Biophys. Acta 750, 1983, pp. 214-216.

46. Holgersson et al., in Koprowski et al. (eds.), Symposium on World'sDebt to Pasteur, pp. 273-301, Alan R. Liss, New York, 1985, in press.

We claim:
 1. A method of inhibiting the interaction between a cellularsecond-step viral binding receptor and a virus which comprises exposingthe virus to a compound which preferentially binds the viral recognitionsite for the binding epitope of said cellular second step viral bindingreceptor, whereby the binding of the virus to the receptor is inhibited,said compound comprising a carbohydrate moiety, a hydrocarbon moiety ofat least five carbon atoms, and an intermediate moiety comprising atleast one sulfur-containing functional group selected from the groupconsisting of --S--, --SO-- and --SO₂ --.
 2. The method of claim 1wherein the intermediate moiety comprises at least one thioetherfunction.
 3. The method of claim 1 wherein the intermediate moietycomprises at least one sulfono function.
 4. A method of inhibiting theinteraction between a cellular second-step viral binding receptor and avirus which comprises exposing the virus to a compound whichpreferentially binds the viral recognition site for the binding epitopeof said cellular second-step viral binding receptor, said compoundcomprising a carbohydrate moiety, a hydrophobic moiety, said hydrophobicmoiety comprising a hydrocarbon moiety having at least one or morechains at least one chain having a length of at least five carbon atoms,and an intermediate polar moiety presenting at least two hydrogenbonding sites.
 5. The method of claim 4 wherein said hydrogen bondingsites are provided by functional groups selected independently from thegroup consisting of amino, carbonyl, hydroxyl, sulfhydryl, sulfoxy orsulfonyl groups.
 6. The method of claim 4 wherein the compound has aconformation substantially similar to that of the natural bindingepitope carried by1-O-beta-D-galactopyransoyl-N-(2-D-hydroxyalkanoyl)-1,3-D-dihydroxy-2-D-aminoalkane.7. The method of claim 4 wherein the carbohydrate moiety comprises oneor more saccharide units, and the saccharide unit nearest theintermediate polar moiety is a pentose, hexose or heptose in furanose orpyranose form.
 8. The method of claim 7 wherein the carbohydrate moietycomprises a plurality of saccharide units including a first saccharideunit nearest the intermediate polar moiety and a second saccharide unitadjacent to the first saccharide unit, the second unit being connectedby a 1→4 linkage to the first unit.
 9. The method of claim 4 wherein thehydrocarbon moiety comprises at least 14 carbon atoms.
 10. The method ofclaim 9 wherein the hydrocarbon moiety comprises 20-30 carbon atoms. 11.The method of claim 4 wherein the compound presents a binding epitopefor the cellular second step viral binding receptor, said epitope havinga length of about 15-20 Å and a width of about 8-10 Å.
 12. The method ofclaim 4 wherein the hydrophobic moiety presents a hydrophobic surfacewith a surface area of at least 50 Å².
 13. The method of claim 4 whereinthe compound has a bent conformation.
 14. The method of claim 8 whereinthe carbohydrate moiety comprises no more than five saccharide units.15. A method of inhibiting the interaction between a cellularsecond-step viral binding receptor and a virus which comprises exposingthe virus to a composition comprising:(1) a compound consistingessentially of (A) a carbohydrate moiety, (B) an intermediate moietycomprising at least two hydrogen bonding sites, and (C) a hydrocarbonmoiety comprising at least 14 carbon atoms, said intermediate moietyconnecting said carbohydrate and hydrocarbon moieties, wherein thesaccharide unit of said carbohydrate moiety which is nearest saidintermediate moiety is a pentose, hexose or heptose in furanose orpyranose form, and if substituted the substituents do not block thealpha side of said nearest saccharide unit, said hydrogen bonding sitesof said intermediate moiety being provided by one or more hydrogenbonding groups selected from the group consisting of amino, carbonyl,hydroxyl, sulfhydryl, sulfoxy or sulfone groups, said compound having atotal length of about 15-20Å, said compound having a bent conformation;said compound specifically binding the viral recognition site for thebinding epitope of said cellular second-step viral binding receptor and(2) a pharmaceutically acceptable vehicle or excipient.